Systems and methods for massively parallel combinatorial analysis of single cells

ABSTRACT

Provided herein are methods that enable parallel evaluation of multiple functional nucleic acids in individual cells or subpopulations of cells, in the context of incubation with other types of single cells. The key insight is concurrent measurement of polynucleic acids derived from small populations of at least two different cell types, such that function in one cell type is linked to the clonal identity of another cell. These methods simultaneously process thousands, millions, or more single cells or small populations of cells. The method integrates molecular, algorithmic, and engineering approaches. This invention has broad and useful application in a number of biological and medical fields, including immunology and drug discovery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/470,836, filed Mar. 13, 2017, which is hereby incorporated in itsentirety by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Mar. 13, 2018, is named39523US_CRF_SequenceListing, and is 34,864 bytes in size.

BACKGROUND OF THE INVENTION

Biological cells are extremely diverse and have an enormous variety ofbiological functions. Functional analysis of cells is therefore afundamental requirement in nearly any biological experiment. Becauseeven genetically homogeneous populations of single cells haveheterogeneous biological functions, biological experiments are bestperformed at the single cell level. However, single cell functionalanalysis is difficult, or impossible, using conventional methods.

Conventionally, functional analysis of “target cells” in response toexposure to “inducer cells” is carried out in tissue culture plates, forexample, 6-well or 96-well plates. Target cells of interest areincubated with an inducer cell type, and then responses of the targetcell are measured by assessing proteins, transcripts, or other kinds ofbiomarkers. Such methods are always carried out on bulk populations,i.e., hundreds, thousands, or millions of target cells are incubatedwith hundreds, thousands, or millions of inducer cells in order todetermine target cell responses to the inducer cells. However, thetarget and inducer cell populations are inherently diverse geneticallyand phenotypically. Even cells with indistinguishable genome sequencesmay react differently to inducer cells, because of epigeneticdifferences, environmental differences, or reasons currently unknown toscience.

Furthermore, methods that are sufficiently sensitive to do functionalassays of a single target or inducer cell have not been available.Typically, quantitative differences in transcript counts between inducedand non-induced cells is only 2-, 5-, or 10-fold, so highly sensitivemethods are required. Similarly, methods that are sufficientlyhigh-throughput to assay millions of single target or inducer cells inparallel have not been available. Additionally, functional analysisoften requires concurrent measurement of transcripts in both the targetand the inducer cells, for example, by concurrently measuring andsequencing transcripts in two cell types. Without such sensitive,high-throughput and combinatorial screening methods, it has been verydifficult to understand functional responses of single target cellsexposed to inducer cells, much less millions of single target or inducercells in parallel.

SUMMARY OF THE INVENTION

The present invention relates to a high-throughput technology that canisolate single target cells with single inducer cells or populations ofinducer cells, combined with a methodology for detecting the response oftarget cells to inducer cells (FIG. 1). In some embodiments, targetcells and inducer cells are additionally incubated with “intermediary”cells, which are a type of induced cell. The present invention providesa highly sensitive method for detecting quantitative differences intranscript counts between induced and non-induced cells that are only2-, 5-, or 10-fold. The present invention further enables acombinatorial measurement, such that diverse populations of target andinducer cells can be analyzed in millions of possible pairwisecombinations. Some methods of the present invention involvequantification of polynucleic acids generated by tethering or linkingpolynucleic acids from more than one cell type. The methods provide anovel way of single cell functional screens that have not been possiblein well-plate methods. The methods further provide the capability totrace functional readout to genetic differences in single target,intermediary, or inducer cells.

One aspect of the present invention relates to a method for functionalanalysis of biological cells, comprising the steps of (1) isolating intoa monodisperse emulsion microdroplet a single target cell from aplurality of target cell clones of a first cell type and one or moreinducer cells from a plurality of inducer cell clones of a second celltype; (2) incubating islolated cells in the monodisperse emulsionmicrodroplet, wherein the isolated cells comprise the single target celland the one or more inducer cells; (3) introducing an aqueous solutioncontaining a lysis reagent into said monodisperse emulsionmicrodroplets, thereby inducing lysis of the isolated cells; (4)capturing RNA released from the isolated cells on a solid surface; and(5) generating a library of hybridized polynucleic acids that comprise atranscript from the isolated cells, wherein the hybridized polynucleicacids are indicative of transcriptional change in the single target cellafter the step of incubating the isolated cells.

In some embodiments, said hybridized polynucleic acids are furtherindicative of transcriptional change in the one or more inducer cellsafter the step of incubating the isolated cells. In some embodiments,said transcriptional change in the one or more inducer cells comprisesincrease of transcripts of a gene by less than tenfold.

In some embodiments, the plurality of target cell clones comprise morethan 10,000 unique cell clones, wherein each target cell clone of theplurality of target cell clones is genetically distinct from each other.In some embodiments, the plurality of inducer cell clones comprise morethan 10,000 unique cell clones, wherein each inducer cell clone of theplurality of inducer cell clones is genetically distinct from eachother. In some embodiments, genetic diversity of the target cell clonesis created by introducing a library of nucleic acid sequences into apopulation of at least 100,000 cells. In some embodiments, geneticdiversity of the inducer cell clones is created by introducing a libraryof nucleic acid sequences into a population of at least 100,000 cells.

In some embodiments, RNA capturing is performed using oligonucleotidesaffixed to bead, each bead has a diameter less than 10 μm.

In some embodiments, the hybridized polynucleic acids are generated byoverlap extension polymerase chain reaction. In some embodiments, thehybridized polynucleic acids are generated by first strand synthesis.

In some embodiments, the first cell type is a library of cells thatexpress T cell receptors. In some embodiments, the first cell type is alibrary of cells that express antibodies. In some embodiments, the firstcell type is a library of cells that express peptide:MHC. In someembodiments, the first cell type is a library of cells that expresspolynucleic acid barcodes.

In some embodiments, cells are isolated into emulsions usingmicrofluidics.

Another aspect of the present invention relates to a compositioncomprising the library of hybridized polynucleic acids. In someembodiments, the composition comprises hybridized polynucleic acids ofat least 10,000 unique sequences. In some embodiments, the compositioncomprises hybridized polynucleic acids of at least 1,000,000 uniquesequences.

Another aspect of the present invention relates to a method forfunctional analysis of a population of cells comprising deep sequencingof the library of hybridized polynucleic acids.

Another aspect of the present invention relates to a compositioncomprising a library of recombinant proteins, generated from thecomposition comprising the library of hybridized polynucleic acids. Insome embodiments, the library of recombinant proteins comprises T cellreceptors. In some embodiments, the library of recombinant proteinscomprises peptide:MHC. In some embodiments, the library of recombinantproteins comprises antibodies.

Another aspect of the present invention relates to a compositioncomprising a first probe and a second probe, wherein (1) the first probecomprises a first subsequence that is complementary to a transcript ofan inducer cell of a first cell type and a second subsequence that iscomplementary to at least a part of the second probe, wherein thetranscript is unique to the first cell type, and (2) the second probecomprises a third subsequence that is complementary to a differenttranscript of a target cell of a second cell type and a fourthsubsequence that is complementary to at least a part of the first probe,wherein the amount of the different transcript changes when the targetcell is incubated with the inducer cell.

In some embodiments, the transcript unique to said first cell typeencodes a T cell receptor. In some embodiments, the transcript unique tosaid first cell type encodes an antibody. In some embodiments, thetranscript unique to said first cell type encodes a peptide:MHC. In someembodiments, the transcript unique to said first cell type encodes apolynucleic acid barcode. In some embodiments, the transcript unique tosaid first cell type encodes a recombinant protein.

Another aspect of the present invention relates to a method for forfunctional analysis of biological cells, comprising the steps of: (1)isolating into a monodisperse emulsion microdroplet a target cell from aplurality of target cell clones of a first cell type and one or moreinducer cells from a plurality of inducer cell clones of a second celltype; (2) incubating isolated cells in the monodisperse emulsionmicrodroplet, wherein the isolated cells comprise the single target celland the one or more inducer cells; (3) isolating RNA from the isolatedcells; (4) generating a library of hybridized polynucleic acids usingthe composition comprising the first probe and the second probe, and (5)deep sequencing the library of hybridized polynucleic acids.

Another aspect of the present invention relates to a method forfunctional analysis of biological cells, comprising the steps of (1)isolating into a monodisperse emulsion microdroplet a single target cellfrom a plurality of target cell clones of a first cell type, one or moreinducer cells from a plurality of inducer cell clones of a second celltype, and one or more intermediary cells from a plurality ofintermediary cell clones of a third cell type; (2) incubating islolatedcells in the monodisperse emulsion microdroplet, wherein the isolatedcells comprise the single target cell, the one or more inducer cells,and the one or more intermediary cells; (3) introducing an aqueoussolution containing a lysis reagent into said monodisperse emulsionmicrodroplets, thereby inducing lysis of the isolated cells; (4)capturing RNA released from the isolated cells on a solid surface; and(5) generating a library of hybridized polynucleic acids that comprise atranscript from the isolated cells, wherein the hybridized polynucleicacids are indicative of transcriptional change in the intermediary cellsafter the step of incubating the isolated cells.

In some embodiments, said hybridized polynucleic acids are indicative oftranscriptional change in the one or more intermediary cells, after thestep of incubating the isolated cells. In some embodiments, saidtranscriptional change in the one or more intermediary cells comprisesincrease of transcripts of a gene by less than tenfold.

In some embodiments, the plurality of target cell clones comprises morethan 10,000 unique cell clones, wherein each target cell clone of theplurality of target cell clones is genetically distinct from the othercell clone of the plurality of cell clones. In some embodiments, theplurality of inducer cell clones comprises more than 10,000 unique cellclones, wherein each inducer cell clone of the plurality of inducer cellclones is genetically distinct from the other cell clone of theplurality of cell clones.

In some embodiments, genetic diversity of the target cell clones iscreated by introducing a library of nucleic acid sequences into apopulation of at least 100,000 cells. In some embodiments, geneticdiversity of the inducer cell clones is created by introducing a libraryof nucleic acid sequences into a population of at least 100,000 cells.

In some embodiments, RNA capturing is performed using oligonucleotidesaffixed to beads, wherein each bead has a diameter less than 10 μm.

In some embodiments, the lysis reagent is a surfactant.

In some embodiments, the hybridized polynucleic acids are generated byoverlap extension polymerase chain reaction. In some embodiments, thehybridized polynucleic acids are generated by first strand synthesis.

In some embodiments, the first cell type is a library of cells thatexpress T cell receptors. In some embodiments, the first cell type is alibrary of cells that express antibodies. In some embodiments, the firstcell type is a library of cells that express peptide:WIC. In someembodiments, the first cell type is a library of cells thattranscriptionally express polynucleic acid barcodes.

In some embodiments, cells are isolated into emulsions usingmicrofluidics.

Another aspect of the present invention relates to a compositioncomprising the library of hybridized polynucleic acids generated by themethod described herein. In some embodiments, the composition compriseshybridized polynucleic acids of at least 1,000, 10,000, 100,000, or1,000,000 unique sequences.

Another aspect of the present invention relates to a method forfunctional analysis of a population of cells by deep sequencing thelibrary of hybridized polynucleic acids generated by the methoddescribed herein.

Another aspect of the present invention relates to a compositioncomprising a library of recombinant proteins, generated from thecomposition comprising the library of hybridized polynucleic acidsgenerated by the method described herein. In some embodiments, thelibrary of recombinant proteins comprises T cell receptors. In someembodiments, the library of recombinant proteins comprises peptide:MHC.In some embodiments, the library of recombinant proteins comprisesantibodies.

Another aspect of the present invention relates to a compositioncomprising a first probe and a second probe, wherein (1) the first probecomprises a first subsequence that is complementary to a transcript ofan inducer cell of a first cell type and a second subsequence that iscomplementary to at least a part of the second probe, wherein thetranscript is unique to the first cell type; and (2) the second probecomprises a third subsequence that is complementary to a differenttranscript of an intermediary cell of a second cell type and a fourthsubsequence that is complementary to at least a part of the first probe,wherein the amount of the different transcript changes when theintermediary cell is incubated with the inducer cell and a target cell.

In some embodiments, the transcript unique to said first cell typeencodes a T cell receptor, an antibody, a peptide:MHC, a polynucleicacid barcode, or a recombinant protein.

Another aspect of the present invention relates to a method forfunctional analysis of biological cells, comprising the steps of (1)isolating into a monodisperse emulsion microdroplet a target cell from aplurality of target cell clones of a first cell type, one or moreinducer cells from a plurality of inducer cell clones of a second celltype and one or more intermediary cells from a plurality of intermediarycell clones of a third cell type; (2) incubating isolated cells in themonodisperse emulsion microdroplet, wherein the isolated cells comprisethe single target cell, the one or more inducer cells, and the one ormore intermediary cells; (3) isolating RNA from the isolated cells; (4)generating a library of hybridized polynucleic acids using thecomposition comprising the first probe and the second probe; and (5)deep sequencing the library of hybridized polynucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic workflow illustrating methods of the presentinvention for parallel functional analysis of single cells.

FIG. 2 shows cell encapsulation in emulsion microdroplets. 1. Channelconstriction. 2. Glass into which microchannels are etched. 3. Cellinput. 4. Lysis/RNA capture bead mix input. 5. Oil input. 6. Emulsionmicrodroplets.

FIG. 3 shows droplet merging for cell lysis. 1. PDMS chip material. 2.Input channel. 3. Cell mixture input. 4. Lysis/bead mixture droplet. 5.Widened channel for droplet fusion. 6. Outlet channel. 7. Electrodes. 8.Fused microdroplet.

FIG. 4 is a diagrammatic workflow of the invention with at least twodifferent single cells, with one clonal inducer cell and one targetcell. 1. Cell mixture encapsulation emulsion microdroplet chip. 2.Clonal inducer cells. 3. Target cells. 4. Clonal inducer cell. 5. Targetcell. 6. Cell culture media inside emulsion microdroplet. 7. Emulsionmicrodroplet fusion chip. 8. Cell mixture emulsion microdroplet. 9.Lysis/RNA capture bead mixture emulsion microdroplet. 10. Transcripttraceable back to clonal inducer cell. 11. Emulsion microdroplet forbinding transcripts to RNA capture beads. 12. Transcript from targetcell, induced by inducer cell. 13. OE-RT-PCR emulsion microdroplet chip.14. RNA-bound bead/OE-RT-PCR mix input. 15. RNA-bound bead/OE-RT-PCR mixinput. 16. Amplicon comprising fusion between cDNA from transcripttraceable back to clonal inducer cell and cDNA from transcript fromtarget cell, induced by inducer cell. 17. OE-RT-PCR mix in emulsionmicrodroplet.

FIG. 5 is a diagrammatic workflow of linking transcripts from at leastthree different single cells, with three cell types, with a target cell,an inducer cell, and an intermediary cell. 1. Cell mixture encapsulationemulsion microdroplet chip. 2. Clonal inducer cells. 3. Target andintermediary cells. 4. Clonal inducer cell. 5. Intermediary cell. 6.Target cell. 7. Cell culture media inside emulsion microdroplet. 8.Emulsion microdroplet fusion chip. 9. Cell mixture emulsionmicrodroplet. 10. Lysis/RNA capture bead mixture emulsion microdroplet.11. Transcript traceable back to clonal inducer cell. 12. Emulsionmicrodroplet for binding transcripts to RNA capture beads. 13.Transcript from target cell, induced by inducer cell. 14. OE-RT-PCRemulsion microdroplet chip. 15. RNA-bound bead/OE-RT-PCR mix input. 16.RNA-bound bead/OE-RT-PCR mix input. 17. Amplicon comprising fusionbetween cDNA from transcript traceable back to clonal inducer cell andcDNA from transcript from target cell, induced by inducer cell. 18.OE-RT-PCR mix in emulsion microdroplet.

FIG. 6 is a diagrammatic workflow of linking transcripts from at leasttwo different single cells, with a target cell and an inducer cell. 1.Inducer clone cell. 2. Target cell. 3. Inducer clone cell transcript. 4.Target cell transcript (induced phenotype, or indicative of inducedtranscriptional change). 5. Inducer clone cell transcript cDNA. 6.OE-RT-PCR linker sequence. 7. Target cell transcript (induced phenotype,or indicative of induced transcriptional change) cDNA. 8. OE-RT-PCRlinker sequence. 9. OE-RT-PCR major, or linked, amplicon; fusion productof target and inducer cell transcript cDNAs. 10. Deep sequencinganalysis of OE-RT-PCR fusion product amplicons. 11. Identification ortrace back of OE-RT-PCR fusion product amplicon sequence to originalinducer cell clone.

FIG. 7 is a diagrammatic workflow of linking transcripts from at leastthree different single cells, with a target cell, an inducer cell, andan intermediary cell. 1. Inducer clone cell. 2. Target cell. 3.Intermediary cell. 4. Action (via a molecule, e.g., a secreted antibody)of inducer cell on intermediary cell. 5. Inducer clone cell transcript.6. Target cell transcript (induced phenotype, or indicative of inducedtranscriptional change). 7. Inducer clone cell transcript cDNA. 8.OE-RT-PCR linker sequence. 9. Target cell transcript (induced phenotype,or indicative of induced transcriptional change) cDNA. 10. OE-RT-PCRlinker sequence. 11. OE-RT-PCR major, or linked, amplicon; fusionproduct of target and inducer cell transcript cDNAs. 12. Deep sequencinganalysis of OE-RT-PCR fusion product amplicons. 13. Identification ortrace back of OE-RT-PCR fusion product amplicon sequence to originalinducer cell clone.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Comprises.” Consists at least of a list of components, i.e.,encompasses all the elements listed, but may also include additional,unnamed elements.

“Cell.” The cell is the basic structural, functional, and biologicalunit of all known living organisms. A cell is the smallest unit of lifethat can replicate independently.

“Transcriptome.” Transcription is the first step of gene expression, inwhich a particular segment of DNA is copied into RNA (especially mRNA)by the enzyme RNA polymerase, to produce “transcripts”. Thesetranscripts have a variety of functions, comprising in particularproviding the basis for translation of proteins inside cells. The“transcriptome” is the complete set of RNA transcripts present in asingle cell or population of cells, or a sampling of transcripts thatessentially comprises the complete set of RNA transcripts present in asingle cell or population of cells.

“Transcriptional change.” A change in the makeup of the transcriptome ofa single cell or population of cells. Said transcriptional change maycomprise a change in 1, 10, 100, 1,000, 10,000, or 100,000 transcripts.In some embodiments of this invention, a transcriptional change leads tochanges in the function of the single cell or population of cells. Insome embodiments of this invention, transcriptional change is induced inresponse to an external stimulus. For example, a T cell binding to itspeptide:MHC antigen target may undergo transcriptional changes thatproduce proteins that lead to adaptive immune functions by the inducedcell. In some embodiments of the invention, transcripts of interest areeither up-regulated or down-regulated.

“Cell phenotype.” A phenotype, or “cell type”, is the composite of acell's observable characteristics or traits, such as its morphology,development, biochemical or physiological properties, behavior, andproducts of behavior. In complex multicellular organisms, cellsspecialize into different cell types that are adapted to particularphenotypes. For avoidance of doubt, phenotype is often synonymous withcell “function”, though changes in cell function do not necessarilyrequire a change in phenotype. In mammals, major cell phenotypes includeskin cells, muscle cells, neurons, T cells, B cells, plasma cells,plasmablasts, fibroblasts, stem cells, and others. Cell types may differboth in appearance and function, yet may be genetically identical. Cellsare able to be of the same genotype (i.e., they are “clonal”) but ofdifferent cell type due to the differential expression of the genes theycontain. Cellular phenotype is the conglomerate of multiple cellularprocesses involving gene and protein expression that result in theelaboration of a cell's particular morphology and function. Many kindsof cells, such as immune cells, undergo phenotypic (i.e., functional)changes in response to external or internal stimuli. For example, memoryB cells mature into plasmablasts upon stimulation with an antigen thatbinds to a B cell receptor on the B cell surface. In certainembodiments, RNA or protein expressed by a cell are used as biomarkersto identify a cell's phenotype.

“Cell clone.” A cell with a unique genetic sequence. For example, two Tcells that share a T cell receptor comprise a cell clone. In otherembodiments, two cells that share an exogenous polynucleic acid barcodecomprise a cell clone. Cell clones may or may not share a cellphenotype. For example, a CD4+ T cell may share a T cell receptorsequence with a CD8+ T cell. In certain embodiments, cell clonescomprise the same cell type.

“Cell population.” A group of cells or cell clones, comprising eithermultiple or single cell phenotypes. In certain embodiments, a cellpopulation comprises 10,000 cell clones of one cell phenotype. Incertain embodiments, a cell population comprises at least 10,000 singlecells of one cell phenotype, wherein thousands of cell clones arepresent. In certain embodiments, a cell population comprises 10,000single cells of 10, 20, 50, or 100 different cell types. For example, atumor comprises millions of cells and dozens of cell types. Cellpopulations may comprise recombinant cells or primary cells.

“Functional analysis.” Functional analysis involves determination orclassification of a cell's function (i.e., phenotype) classicallythrough experimental methods such as transcript expression analysis(e.g., quantitative PCR, DNA microarrays, RNA-sequencing), genomesequencing or genotyping (e.g., immune repertoire sequencing,quantitative PCR, whole genome shotgun sequencing), protein expressionanalysis (e.g., flow cytometry, ELISA), measurement of glycans (e.g.,mass spectrometry), or measurement of any molecule that is a hallmark ofthe function of the cell. Because cellular function can be plastic,i.e., cellular phenotype can change in response to external stimuli,measurement of cellular function is particularly useful in screening fordrugs or molecules that induce a specific biological function via cellfunctional changes. For avoidance of doubt, functional analysis isgenerally synonymous with phenotype analysis, although changes in cellfunction do not necessarily require a change in phenotype.

“Library.” A pool of at least two polynucleic acids, cell clones,molecules, or proteins. In certain embodiments, a library is used toscreen for biologically active proteins. In other embodiments, a libraryof cell clones is mixed with a drug, and then a biological assay is usedto discern which cell clones are responsive to the drug. In otherembodiments, a library of drugs is mixed with a single cell clone, andthen a biological assay is used to discern which drugs cause a responsein the cell clone. A library may comprise 100, 1,000, 10,000, 100,000,or 1 million different peptide:MHC targets, either as a polynucleotidelibrary that codes for the peptide:MHC targets, or as cells engineeredto express the peptide:MHC. In other embodiments, a library comprises100, 1,000, 10,000, 100,000, or 1 million polynucleic acid barcodes, orcells engineered to express the polynucleic acid barcodes as RNA.

“Combinatorial.” Relating to combinations of libraries of cells,proteins, polynucleic acids, or other types of molecules. Acombinatorial functional analysis involves determining the function ofrandom combinatorial pairs of components from such libraries. Becausethe components of the libraries are paired randomly, the number ofpossible combinations is the size of the first library multiplied by thesize of the second library. For example, a library of 100 clonesscreened combinatorially against a library of 1,000 clones results in100,000 theoretical combinations. Combinatorial functional analysis isuseful for discovery of novel molecules or cellular interactions thatinduce cell functions of interest. In certain embodiments of the presentinvention, a genetically diverse library of cell clones iscombinatorially screened against another diverse (for example, 100,1,000, 10,000, 100,000, or 1 million clones) library of cell clones. Incertain embodiments of the invention, a diverse library of cell clonesis combinatorially screened against an oligoclonal (for example, fewerthan 10) library of cell clones.

“Polynucleic acid.” A polynucleic acid is a double or single strandedmolecule of RNA or DNA, typically comprising 5, 10, 20, 50, 100, 1,000,10,000, or more base pairs. Polynucleic acids may be synthetic, i.e.,manufactured chemically from individual nucleotides, amplified, i.e.,generated enzymatically from template nucleic acids using a polymerase,or purified from biological systems, i.e., extracted from cells or otherbiological materials. Polynucleic acids derived from, or detected in,biological cells, often serve as “biomarkers” that indicate functionaldifferences between cells or populations of cells. Polynucleic acidshave many sub-categories familiar to those skilled in the art.Complementary DNA, or cDNA, is DNA synthesized by using an enzyme suchas reverse transcriptase to make cDNA from an RNA template. An“oligonucleotide” is a short (6-100 nucleotides) single stranded DNA orRNA sequence, typically manufactured synthetically by a commercialprovider such as IDT DNA or ThermoFisher.

“Variable immune receptor.” A variable immune receptor is anyglycoprotein or glycoprotein complex that varies from cell to cell, orperson to person. Variable immune receptors comprise critical innate andadaptive immune diversity required to identify invasive (or pathogenic)cells, viruses, bacteria, or other biologic material. In certainembodiments, an immune receptor that comprises the adaptive immunesystem, for example, an antibody or a T cell receptor. Most adult humansexpress billions of such variable receptors, in billions of different Tcells or B cells. In other embodiments, an immune receptor thatcomprises immune system components that vary from individual toindividual, for example, MHC or killer cell immunoglobulin-like (KIR)receptors.

“T cell receptor.” The T cell receptor, or TCR, is a molecule found onthe surface of T cells, or T lymphocytes, that are responsible forrecognizing fragments of antigen as peptides bound to majorhistocompatibility complex (MHC) molecules. The TCR is adisulfide-linked membrane-anchored heterodimeric protein normallyconsisting of the highly variable alpha (α) and beta (β) chainsexpressed as part of a complex with the invariant CD3 chain molecules. Tcells expressing this receptor are referred to as α/β (or αβ) T cells,though a minority of T cells express an alternate receptor, formed byvariable gamma (γ) and delta (δ) chains, referred as γδ T cells. Eachchain is composed of two extracellular domains: Variable (V) region anda Constant (C) region, both of Immunoglobulin superfamily domain formingantiparallel beta-sheets. The Constant region is proximal to the cellmembrane, followed by a transmembrane region and a short cytoplasmictail, while the Variable region binds to the peptide:MHC complex. Thevariable domain of both the TCR α-chain and β-chain each have threehypervariable or complementarity determining regions (CDRs), whereas thevariable region of the β-chain has an additional area ofhypervariability (HV4) that does not normally contact antigen and,therefore, is not considered a CDR. The residues are located in tworegions of the TCR, at the interface of the α- and β-chains and in theβ-chain framework region that is thought to be in proximity to the CD3signal-transduction complex. CDR3 is the main CDR responsible forrecognizing processed antigen, although CDR1 of the alpha chain has alsobeen shown to interact with the N-terminal part of the antigenicpeptide, whereas CDR1 of the β-chain interacts with the C-terminal partof the peptide. CDR2 is thought to recognize the MHC. CDR4 of the(3-chain is not thought to participate in antigen recognition, but hasbeen shown to interact with superantigens. The constant domain of theTCR domain consists of short connecting sequences in which a cysteineresidue forms disulfide bonds, which forms a link between the twochains. Each recombined TCR possess unique antigen specificity,determined by the structure of the antigen-binding site formed by the αand β chains in case of αβ T cells or γ and δ chains on case of γδ Tcells. It is based mainly on genetic recombination of the DNA encodedsegments in individual somatic T cells—either somatic V(D)Jrecombination using RAG1 and RAG2 recombinases or gene conversion usingcytidine deaminases. The intersection of these specific regions (V and Jfor the alpha or gamma chain; V, D, and J for the beta or delta chain)corresponds to the CDR3 region that is important for peptide:MHCrecognition. For avoidance of doubt, the term “TCR” throughout thisdisclosure embodies the full variety of possible recombinant derivativeformats, and could be derived from any animal with an adaptive immunesystem, such as a human, mouse, camel, cow, bird, or fish. TCRs can beengineered into soluble form, for example by engineering chimeras withCD3 or Fc protein domains. These soluble TCRs then act as drugs byactivating or antagonizing molecular targets of relevance to disease,for example, cancer.

“T cell.” A T cell is a lymphocyte of a type produced or processed bythe thymus gland and actively participating in the immune response. Tcells play a central role in cell-mediated immunity. T cells can bedistinguished from other lymphocytes, such as B cells and natural killercells, by the presence of a T-cell receptor on the cell surface. Theseveral subsets of T cells each have a distinct function. T helper cells(T_(H) cells) assist other white blood cells in immunologic processes,including maturation of B cells into plasma cells and memory B cells,and activation of cytotoxic T cells and macrophages. These cells arealso known as CD4+ T cells because they express the CD4 glycoprotein ontheir surfaces. Helper T cells become activated when they are presentedwith peptide antigens by MHC class II molecules, which are expressed onthe surface of antigen-presenting cells (APCs). Once activated, theydivide rapidly and secrete small proteins called cytokines that regulateor assist in the active immune response. These cells can differentiateinto one of several subtypes, including TH1, TH2, TH3, TH17, TH9, orTFH, which secrete different cytokines to facilitate different types ofimmune responses. Signaling from the APC directs T cells into particularsubtypes. Cytotoxic T cells (Tc cells, CTLs, T-killer cells, killer Tcells) destroy virus-infected cells and tumor cells, and are alsoimplicated in transplant rejection. These cells are also known as CD8+ Tcells since they express the CD8 glycoprotein at their surfaces. Thesecells recognize their targets by binding to antigen associated with MHCclass I molecules, which are present on the surface of all nucleatedcells. Through IL-10, adenosine, and other molecules secreted byregulatory T cells, the CD8+ cells can be inactivated to an anergicstate, which prevents autoimmune diseases. Memory T cells are a subsetof antigen-specific T cells that persist long-term after an infectionhas resolved. They quickly expand to large numbers of effector T cellsupon re-exposure to their cognate antigen, thus providing the immunesystem with “memory” against past infections. Regulatory T cells(suppressor T cells) are crucial for the maintenance of immunologicaltolerance. Their major role is to shut down T cell-mediated immunitytoward the end of an immune reaction and to suppress autoreactive Tcells that escaped the process of negative selection in the thymus.Suppressor T cells along with Helper T cells can collectively be calledRegulatory T cells due to their regulatory functions. Two major classesof CD4+ Treg cells have been described—FOXP330 Treg cells and FOXP3−Treg cells. The majority of human T cells rearrange their alpha and betachains on the cell receptor and are termed alpha beta T cells (ab Tcells) and are part of the adaptive immune system. Specialized gammadelta T cells, (a small minority of T cells in the human body, morefrequent in ruminants), have invariant T cell receptors with limiteddiversity, that can effectively present antigens to other T cells andare considered to be part of the innate immune system. The geneticrearrangements and mutations that lead to TCR expression produces a Tcell “clone”. When the TCR engages with antigenic peptide and MHC(peptide:MHC), the T lymphocyte is activated through signaltransduction, that is, a series of biochemical events mediated byassociated enzymes, co-receptors, specialized adaptor molecules, andactivated or released transcription factors. Immortal cell lines areoften used experimentally to study T cell function, for example, theJurkat cell line. In some embodiments of the invention, the TCRabexpressed by Jurkat is knocked out, or deactivated, and a recombinantTCRab is introduced into the genome or transiently expressed through anexpression construct. T cells are engineered into “cellulartherapeutics” by introducing recombinant TCR constructs, for examplethrough lentivirus transduction. T cell therapeutics are allogeneic orautologous, and are used to treat cancer and other kinds of seriousdisease. The engineered TCR is therefore a kind of drug that acts via aT cell.

“Antigen.” The other member of a cognate pair for an antibody or T cellreceptor. In certain embodiments, antibodies or T cell receptorsspecifically bind to a single antigen. In other embodiments, antibodiesor T cell receptors bind to multiple antigens. Antibodies typically bindto proteins or glycoproteins in their native conformation, whereas Tcell receptors require processed peptide antigens presented on thesurface of an antigen presenting cell by an MHC. In certain embodiments,antigens are soluble, whereas in other embodiments, antigens aretethered to the surface of a cell.

“Antigen presenting cell.” An antigen presenting cell (APC) displays anantigen peptide on its cell membrane. Antigen peptides are the productof proteolytic processing inside the APC. The antigenic peptides arethen bound to a major histocompatibility complex (MHC) protein on thecell membrane of the APC. The bound complex is known as the peptide:MHCcomplex. T cell receptors do not bind antigen peptides directly, butinstead require a peptide:MHC complex. In some embodiments, the peptideis derived from full proteins expressed by the APC. In otherembodiments, the peptide is derived from viral proteins, and display ofthe viral-derived peptide is a hallmark of a cell infected by a virus.In certain embodiments, at least one plasmid encoding a full protein,partial protein, or polypeptide is introduced into a cell, and theplasmid drives expression of a recombinant peptide:MHC on the surface ofthe APC. In certain embodiments, APCs are incubated with peptides,peptide mixes, or proteins, resulting in a peptide:MHC on the APCmembrane surface. In certain embodiments, cellular assays are performedwith APCs. In certain embodiments, cellular assays are performed withAPCs that are immortal cell lines (e.g., T2 cells), or primary cells(e.g., B cells).

“Antibody.” An antibody (Ab), also known as an immunoglobulin (Ig), is alarge, Y-shaped protein produced mainly by plasma cells that is used bythe immune system to neutralize pathogens such as bacteria and viruses.The antibody recognizes a unique molecule of the harmful agent, calledan antigen, via the Fab's variable region. Each tip of the “Y” of anantibody contains a paratope (analogous to a lock) that is specific forone particular epitope (similarly analogous to a key) on an antigen,allowing these two structures to bind together with precision. Usingthis binding mechanism, an antibody can tag a microbe or an infectedcell for attack by other parts of the immune system, or can neutralizeits target directly (for example, by blocking a part of a microbe thatis essential for its invasion and survival). Depending on the antigen,the binding may impede the biological process causing the disease or mayactivate macrophages to destroy the foreign substance. The ability of anantibody to communicate with the other components of the immune systemis mediated via its Fc region (located at the base of the “Y”), whichcontains a conserved glycosylation site involved in these interactions.The production of antibodies is the main function of the humoral immunesystem. Antibodies can occur in two physical forms, a soluble form thatis secreted from the cell to be free in the blood plasma, and amembrane-bound form that is attached to the surface of a B cell and isreferred to as the B-cell receptor (BCR). The BCR is found only on thesurface of B cells and facilitates the activation of these cells andtheir subsequent differentiation into either antibody factories calledplasma cells or memory B cells that will survive in the body andremember that same antigen so the B cells can respond faster upon futureexposure. In most cases, interaction of the B cell with a T helper cellis necessary to produce full activation of the B cell and, therefore,antibody generation following antigen binding. Soluble antibodies arereleased into the blood and tissue fluids, as well as many secretions tocontinue to survey for invading microorganisms. They are typically madeof basic structural units—each with two large heavy chains and two smalllight chains. There are several different types of antibody heavy chainsthat define the five different types of crystallisable fragments (Fc)that may be attached to the antigen-binding fragments. The fivedifferent types of Fc regions allow antibodies to be grouped into fiveisotypes. Each Fc region of a particular antibody isotype is able tobind to its specific Fc Receptor (except for IgD, which is essentiallythe BCR), thus allowing the antigen-antibody complex to mediatedifferent roles depending on which FcR it binds. The ability of anantibody to bind to its corresponding FcR is further modulated by thestructure of the glycan(s) present at conserved sites within its Fcregion. The ability of antibodies to bind to FcRs helps to direct theappropriate immune response for each different type of foreign objectthey encounter. Though the general structure of all antibodies is verysimilar, a small region at the tip of the protein is extremely variable,allowing millions of antibodies with slightly different tip structures,or antigen-binding sites, to exist. This region is known as thehypervariable region. Each of these variants can bind to a differentantigen. This enormous diversity of antibody paratopes on theantigen-binding fragments allows the immune system to recognize anequally wide variety of antigens. The large and diverse population ofantibody paratope is generated by random recombination events of a setof gene segments that encode different antigen-binding sites (orparatopes), followed by random mutations in this area of the antibodygene, which create further diversity. This recombinatorial process thatproduces clonal antibody paratope diversity is called V(D)J or VJrecombination. Basically, the antibody paratope is polygenic, made up ofthree genes, V, D, and J. Each paratope locus is also polymorphic, suchthat during antibody production, one allele of V, one of D, and one of Jis chosen. These gene segments are then joined together using randomgenetic recombination to produce the paratope. The regions where thegenes are randomly recombined together is the hypervariable region usedto recognize different antigens on a clonal basis. Soluble antibodiesare commonly used as therapeutic drugs, for example, rituximab,adalimumab, pembrolizumab, or trastuzumab. Antibodies are sometimesreformatted as Single Chain Fragment Variable (scFv), comprising a heavyand light chain fused together as a single protein, via a peptidelinker. In some scenarios, scFv are reformatted as Chimeric AntigenReceptors (CARs), which are then engineered into T cells to createcellular therapeutics called CAR-Ts. For avoidance of doubt, the term“antibodies” throughout this disclosure embodies the full variety ofpossible recombinant derivative formats, and could be derived from anyanimal with an adaptive immune system, such as a human, mouse, camel,cow, bird, or fish.

“Natural killer cell.” Natural killer cells (also known as NK cells, Kcells, and killer cells) are a type of lymphocyte (a white blood cell)and a component of innate immune system. NK cells play a major role inthe host-rejection of both tumors and virally infected cells. Typically,immune cells detect major histocompatibility complex (MHC) presented oninfected cell surfaces, triggering cytokine release, causing lysis orapoptosis. NK cells are unique, however, as they have the ability torecognize stressed cells in the absence of antibodies and MHC, allowingfor a much faster immune reaction. They were named “natural killers”because of the initial notion that they do not require activation tokill cells that are missing “self” markers of MHC class 1. This role isespecially important because harmful cells that are missing MHC Imarkers cannot be detected and destroyed by other immune cells, such asT lymphocyte cells. NK cells also kill cells by a mechanism calledAntibody-Dependent Cell-mediated Cytotoxicity (ADCC), which starts withsoluble antibodies binding to antigens on a target cell's surface.Antibodies that bind to antigens can be recognized by FcgRIII (CD16)receptors expressed on NK cells, resulting in NK activation, release ofcytolytic granules and consequent cell apoptosis. This is a major cellkilling mechanism of some monoclonal antibodies like rituximab,ofatumumab, and others. In certain embodiments, a cell line such as theNK-92 cell line is used in place of primary NK cells.

“Target.” A biological molecule to which a drug binds in order to inducea pharmacological function. In certain embodiments, the target is aprotein produced by a cell and expressed on the cell membrane. Targetsalso comprise nucleic acids, lipids, glycans, and glycoproteins. Incertain embodiments, the target is an antigen, for example, a proteinrecognized by an antibody or a peptide:MHC recognized by a TCR.

“Target cell.” A biological cell that expresses an antigen or target. Incertain embodiments of the invention, the target or antigen is bound tothe cell membrane of the target cell, and therefore exposed to theextracellular space. In certain embodiments of this invention, thetarget cell undergoes quantifiable changes in 1, 10, 100, or 10,000 mRNAtranscripts as a result of the inducer cell interacting with the antigenor target on the surface of the target cell. In some embodiments of theinvention, the quantifiable changes in the target cell are endogenoustranscripts. In some embodiments of the invention, the quantifiablechanges in the target cell are transcripts arising from recombinantlyengineered “reporter” constructs that have been introduced into thetarget cell. In some embodiments of the invention, the reporterconstructs contain promoters, enhancers, or other regulatory elementsthat induce transcription upon contact with signals resulting from theinducer cell contacting the target cell. In some embodiments of theinvention, transcripts of interest are either up-regulated ordown-regulated.

“Inducer cell.” A biological cell that expresses a ligand or inducermolecule that binds to an antigen or target on the target cell. Incertain embodiments of the present invention, the inducer cell secretesproteins or molecules that then bind to the target cell to inducequantifiable transcriptional changes. In other embodiments of theinvention, proteins or molecules on the inducer cell surface bind to thetarget cell to induce quantifiable transcriptional changes. In certainembodiments, the inducing proteins or molecules comprise a singlespecies, whereas in other embodiments of the invention, the inducingproteins or molecules comprise 2, 5, 10, 100, or 1,000 individualspecies. In certain embodiments of this invention, the inducer cellundergoes quantifiable changes in 1, 10, 100, or 10,000 mRNA transcriptsas a result of the inducer cell interacting with the antigen or targeton the surface of the target cell.

“Intermediary cell.” A biological cell that responds functionally to theinteraction between an inducer and a target cell, or to the interactionbetween a protein secreted by an inducer cell and proteins expressed bya target cell. In certain embodiments of this invention, theintermediary cell undergoes quantifiable changes in 1, 10, 100, or10,000 mRNA transcripts as a result of the inducer cell interacting withthe antigen or target on the surface of the target cell. In otherembodiments of the invention, proteins or molecules secreted by theinducer cell surface bind to the target cell to induce quantifiabletranscriptional changes in the intermediary cells. In some embodimentsof the invention, the quantifiable changes in the intermediary cell aretranscripts arising from recombinantly engineered “reporter” constructsthat have been introduced into the intermediary cell.

“Synthetic polynucleic acid.” Chemically or enzymatically synthesizedRNA or DNA. To synthesize single-stranded RNA or DNA, or“oligonucleotides”, the chemical synthesis process can be implemented assolid-phase synthesis using phosphoramidite method and phosphoramiditebuilding blocks derived from protected 2′-deoxynucleosides (dA, dC, dG,and dT), ribonucleosides (A, C, G, and U), or chemically modifiednucleosides, e.g. LNA or BNA. To obtain the desired oligonucleotide, thechemical building blocks can be sequentially coupled to the growingoligonucleotide chain in the order required by the sequence of theproduct. Typically, synthetic oligonucleotides are single-stranded DNAor RNA molecules around 15-25 bases in length. Synthetic polynucleicacids can be also generated by enzymatic methods, such as reversetranscription (RT), polymerase chain reaction (PCR), Gibson assembly,overlap extension PCR (OE-PCR), overlap extension RT-PCR (OE-RT-PCR),emulsion PCR, emulsion RT-PCR, emulsion OE-RT-PCR, emulsion OE-PCR,ligase chain reaction (LCR), hybridization, in vitro transcription, orany other cell-free molecular biological method that makes use ofpurified enzymes.

“Polynucleic acid barcode.” A polynucleic acid barcode comprises asynthetic polynucleic acid that enables an experimentalist to identify acell clone, i.e., a unique identifier. In some embodiments, barcodes areengineered into the genome of a cell, contained within an expressionplasmid, or encoded into a recombinant or synthetic RNA sequence. Insome embodiments, a barcode is attached to a solid surface, such as aone micron diameter magnetic bead. In some embodiments, populations ofclones contain 10, 100, 1,000, 10,000, 100,000, or 1 million differentbarcodes. The barcodes can be sequenced through bulk sequencing,enabling high throughput combinatorial analysis of cell function.

“Reverse transcription.” The process by which a reverse transcriptase(RT) enzyme is used to generate complementary DNA (cDNA) from an RNAtemplate. Reverse transcriptase is commonly used in research to applythe polymerase chain reaction technique to RNA in a technique calledreverse transcription polymerase chain reaction (RT-PCR). The classicalPCR technique can be applied only to DNA strands, but, with the help ofreverse transcriptase, RNA can be reverse transcribed into DNA, thusmaking PCR analysis of RNA molecules possible. Reverse transcriptase isused also to create cDNA libraries from mRNA.

“Polymerase chain reaction.” Polymerase chain reaction (PCR) is atechnique used in molecular biology to amplify a single copy or a fewcopies of a piece of DNA across several orders of magnitude, generatingthousands to millions of copies of a particular DNA sequence. The methodrelies on thermal cycling, consisting of cycles of repeated heating andcooling of the reaction for DNA melting and enzymatic replication of theDNA. Primers (short DNA fragments) containing sequences complementary tothe target region along with a DNA polymerase, which the method is namedafter, are key components to enable selective and repeatedamplification. As PCR progresses, the DNA generated is itself used as atemplate for replication, setting in motion a chain reaction in whichthe DNA template is exponentially amplified. PCR can be extensivelymodified to perform a wide array of genetic manipulations. PCR is notgenerally considered to be a recombinant DNA method, as it does notinvolve cutting and pasting DNA, only amplification of existingsequences. Almost all PCR applications employ a heat-stable DNApolymerase, such as Taq polymerase (an enzyme originally isolated fromthe bacterium Thermus aquaticus). This DNA polymerase enzymaticallyassembles a new DNA strand from DNA building-blocks, the nucleotides, byusing single-stranded DNA as a template and DNA oligonucleotides (alsocalled DNA primers), which are required for initiation of DNA synthesis.The vast majority of PCR methods use thermal cycling, i.e., alternatelyheating and cooling the PCR sample through a defined series oftemperature steps. In the first step, the two strands of the DNA doublehelix are physically separated at a high temperature in a process calledDNA melting. In the second step, the temperature is lowered and the twoDNA strands become templates for DNA polymerase to selectively amplifythe target DNA. The selectivity of PCR results from the use of primersthat are complementary to the DNA region targeted for amplificationunder specific thermal cycling conditions.

“Hybridization.” Any process whereby two polynucleic acids are fused toform a single polynucleic acid molecule. Hybridization can occur by anyprocess, natural or artificial, that results in two single-strandedpolynucleic acids forming base pairing that result in a molecule that isat least partially double stranded. Base pairings conventionally occurthrough reverse complementarity, for example, guanine-cytosine,adenine-thymine, or adenine-uracil. In some embodiments, the hybridizedbase pairs are adjacent, for example, two single-stranded polynucleicacids that are each 100 nucleotides comprise 20 nucleotide subsequencesthat are reverse complements. Under the proper conditions, the twopolynucleic acids would hybridize across these complementary nucleotidesubsequences, forming a hybridized molecule. The amplification processcalled “overlap extension PCR” generates a plurality of fused, doublestranded DNA products that result from the initial hybridization stepbetween two polynucleotides that comprise complementary nucleotidesubsequences.

“Microfluidics.” Microfluidics is the science and technology ofmanipulating and controlling fluids, usually in the range of microliters(10⁻⁶) to picoliters (10⁻¹²), in networks of channels with lowestdimensions from tens to hundreds micrometers. Typically, fluids aremoved, mixed, separated or otherwise processed. Numerous applicationsemploy passive fluid control techniques like capillary forces. In someapplications, external actuation means are additionally used for adirected transport of the media. Examples are rotary drives applyingcentrifugal forces for the fluid transport on the passive chips. Activemicrofluidics refers to the defined manipulation of the working fluid byactive (micro) components such as micropumps or microvalves. Micropumpssupply fluids in a continuous manner can be used for dosing. Microvalvescan determine the flow direction or the mode of movement of pumpedliquids. Processes which are normally carried out in a lab can beminiaturized on a single chip in order to enhance efficiency andmobility as well as to reduce sample and reagent volumes. Droplet-basedmicrofluidics as a subcategory of microfluidics in contrast withcontinuous microfluidics has the distinction of manipulating discretevolumes of fluids in immiscible phases with low Reynolds number andlaminar flow regimes. Two immiscible phases used for the dropletgeneration are termed as the continuous phase (medium in which dropletsare generated) and dispersed phase (the droplet phase). The size of thegenerated droplets is mainly controlled by the flow rates of thecontinuous phase and dispersed phase, interfacial tension between twophases and the geometry used for the droplet generation.

“Microdroplet.” A spherical, small volume of liquid, typically withvolume less than one microliter. Microdroplets comprise aqueous-in-oilmicrodroplets and oil-in-aqueous microdroplets. A population ofaqueous-in-oil microdroplets or oil-in-aqueous microdroplets comprise an“emulsion”. Emulsions can be monodisperse, e.g., comprisingmicrodroplets substantially the same volume, for example, varying by nomore than 25% in diameter, or polydisperse, e.g., comprisingmicrodroplets of a variety of volumes, for example, varying by >25% indiameter. Microdroplets are a means for performing high-throughputmolecular, cellular, or biochemical experiments. Microdroplets serve topartition liquid reactions and therefore serve a similar function as aphysical container. Millions or billions of microdroplets can bedeposited in a small (for example, one milliliter) physical container,enabling very large combinatorial screening on single cells. In someembodiments of the present invention, monodisperse microdroplets aregenerated using microfluidics, i.e., “droplet microfluidics”. In otherembodiments of the invention, polydisperse microdroplets are generatedusing a shaking or mixing apparatus.

“Physical container.” Physical containers used in molecular biology,cell biology, or biochemistry refer to tubes, plates, dishes, vials, orother formats comprising solid plastic, glass, polymer, or other solidmaterial. In some embodiments, the physical container is inert, i.e.,the container serves only to physically contain liquids for a molecular,cellular, or biochemical experiment. In some embodiments, reactivecells, molecules, proteins, drugs, or biochemical container are affixedto the physical container. Physical containers are a means forperforming molecular, cellular, or biochemical experiments. To increaseprocessing throughput, physical containers can be used together withrobotic systems. In some embodiments, throughput is increased by usingmicrofluidic chips that comprise physical containers, for example,nanoliter chambers on a glass, plastic, or PDMS microfluidic chip.

“Solid support.” Solid supports used in molecular biology, cell biology,or biochemistry refer to beads or other geometric formats comprisingsolid plastic, glass, polymer, or other solid material. In someembodiments of the invention, reactive cells, polynucleic acids,proteins, or other molecules are affixed to solid supports. The solidsupports are then introduced into a physical container or microdroplet,such that a biochemical, cellular, or molecular function is enabled. Thesolid supports can then be washed, or removed, simplifying multi-steplaboratory processes. In some embodiments, the solid supports aremagnetic beads of one, ten, or one hundred microns. In some embodiments,synthetic polynucleic acids are affixed to the magnetic beads, enablingpurification of endogenous cellular polynucleic acids that arecomplementary to the synthetic polynucleic acids, also called “probes”.In some embodiments, solid supports are beads coated with antibodies,which are then used to purify cells that express antigens with affinityfor the antibodies.

“Bulk sequencing.” Synonymous with deep sequencing, ultra-highthroughput sequencing, massively parallel sequencing, andnext-generation sequencing. Bulk sequencing comprises obtaining hundredsof thousands, millions, hundreds of millions, or billions of DNAsequence reads in parallel. In many embodiments, a diverse library ofDNA is generated using methods such as PCR, RT-PCR, or hybridization andthen a plurality of the library is sequenced using bulk sequencing.Methods can comprise sequencing by synthesis, nanopore sequencing, andpyrosequencing. As of 2017, commercial providers of bulk sequencingcomprise Illumina, Pacific Biosciences, Oxford Nanopore, and Roche.

Overview of the Invention

One aspect of the present invention relates to concurrent measurement ofpolynucleic acids derived from at least two different cell types. Themeasurement can be performed in a massively parallel fashion on a smallnumber of cells, or combinatorial screens can be performed on millionsof different cell type combinations. In som embodiments, cells arecombinatorially isolated into reaction containers, incubated to induce abiological response, and lysed to isolate RNA while retaining thecombinatorial context. Transcripts from at least two different celltypes can be physically linked by hybridization, and then the linkedclones can be subject to deep sequencing on a massively parallel scale(FIG. 1).

The methods can involve isolation of single cells or subpopulations ofcells into microemulsion droplets, gels, or microfluidic reactioncontainers. Millions of cells can be isolated or compartmentalized in amassively parallel manner to generate cell mixtures that representgenetically distinct pairwise combinations (FIG. 2).

The cell mixtures can comprise one or more target cells, one or moreinducer cells, and/or one or more intermediary cells. The target cellscan comprise populations of homogeneous cells or genetically distinctclones (for example, B cells, T cells, cells engineered with barcodes,cells engineered to express peptide antigens, primary cancer cells insingle cell suspension). The inducer cells can comprise populations ofhomogenous cells or genetically distinct clones (for example, B cells, Tcells, cells engineered with barcodes, cells engineered to expresspeptide antigens, NK cells). In some embodiments, intermediary cells areused, and the intermediary cells can comprise populations of homogeneouscells or genetically distinct clones (for example, NK cells).

In some embodiments, the target cells and inducer cells are mixed with alibrary of polynucleic acid barcodes affixed to a solid support (forexample, beads, or a protein). In some embodiments, the cell mixturesare additionally incubated in the same microemulsion droplets, gels, orreaction containers with a stimulus, for example, a homogeneouspopulation of cells, a library of reagents, or a single reagent.

The mixtures of cells can be then lysed by introducing a reagent intothe microemulsion droplets, gels, or microfluidic reaction containers.In some embodiments, this step comprises fusing microemulsion dropletscontaining the cells with microemulsion droplets containing the lysisreagent, thus preserving the compartmentalization of the cell mixtures(FIG. 3). After lysis, transcripts from the cell mixtures can bepurified, for example, using beads coated with oligo-dToligonucleotides.

In some embodiments, two or more polynucleotide targets are hybridized,such that polynucleic acids that differentiate clones are linked to RNAtranscripts that indicate functional changes (FIGS. 4-5). The keyinsight is to fuse transcripts derived from at least two different celltypes, for example, antibody target encoding transcripts andantibody-encoding transcripts derived from antibody-producing cells(wherein antibody-producing cells are the inducer cells). The hybridizedpolynucleic acid molecules can be then sequenced by bulk, orhigh-throughput, sequencing. Any high-throughput sequencing method knownin the art can be employed.

The bulk sequencing data can be subsequently analyzed algorithmically todetermine which clones from the initial clone library demonstrate afunctional change in response to the inducer cell stimulus, or stimuli(FIGS. 6-7). Sequencing of hybridized nucleic acid molecules frommultiple cell types enables concurrent measurement of at least onetranscript from each of at least two cell types, for example, anantibody target producing cell and an antibody-producing cell. Becauseof the extreme sensitivity of deep sequencing, transcript counts thatare only 2-, 5-, or 10-fold different between induced and non-inducedcells are detectable. Therefore, the method of the present invention canprovide insight into the functional response of single target cellsexposed to inducer cells, across millions of single target and inducercells in parallel, and enables combinatorial functional screens thathave never before been possible. In some embodiments, the hybridizedpolynucleic acids are further used to make libraries of recombinantproteins, which can be subsequently further screened for binding orfunction.

Provided herein are detailed descriptions of methods of the invention.Also provided herein are detailed descriptions of examples ofembodiments of the invention, with particular application to immunology,drug discovery, drug development, and cancer biology.

Other Interpretational Conventions

Ranges recited herein are understood to be shorthand for all of thevalues within the range, inclusive of the recited endpoints. Forexample, a range of 1 to 50 is understood to include any number,combination of numbers, or sub-range from the group consisting of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

Methods of the Invention

1) Generation of DNA Libraries

Some embodiments of the present invention involves generation oflibraries of antibody clones by isolating B cells from mammalian donors,and then fusing the primary cells with myeloma cells, using techniquessuch as electrofusion, which are well known to those skilled in the art(Smith & Crowe, Microbiol Spectr. 2015 3(1): AID-0027-2014). Theresulting cells, known as hybridomas, can be easier to rear in culturethan primary cells. A variety of methods have been used to make T and Bcell hybridomas, using primary cells from species comprising mice andhumans. Using these methods, libraries of tens of thousands, hundreds ofthousands, or millions of clones of cells that each express a unique TCRor antibody can be made.

Some embodiments of the present invention relate to methods ofgenerating DNA libraries of a gene by isolating RNA from primary cells,for example, a tumor, a liver, a brain, blood, bone marrow, peripherialblood mononuclear cells, muscle tissue, cerebrospinal fluid, kidneytissue, lung lavage, lung tissue, immortal cell lines, skin tissue, orany other tissue or cell type. Reverse transcriptase can be used tosynthesize cDNA from the RNA. For example, RNA is incubated with M-MuLVRT at 42° C. with an oligo-dT primer for one hour. In some embodiments,the oligo-dT primer is fused with a nucleic acid barcode sequence,flanked by universal amplification primers, which enables specificamplification of the barcode and trace back of the barcode to a cDNAsequence. This enables de-multiplexing of complex mixtures of clones.RT-based methods have the advantage of cheaply and quickly generatingDNA libraries comprised of tens of thousands, hundreds of thousands, ormillions of DNA clones in parallel. To recover a plurality of cDNAclones of interest, the full cDNA library can be subjected to PCR usinga reaction comprising gene-specific primers, a thermostable polymerasesuch as Taq, and thermocycling consisting of denaturation (95° C. for 30seconds), 30 cycles of amplification (95° C. for 15 seconds, 62° C. for60 seconds, and 68° C. for 3 minutes), followed by a final extension at68° C. for 5 minutes.

Some embodiments of the present invention relate to a method ofgenerating DNA libraries of antibodies, TCRs, or any other kind ofgenetic sequence by DNA synthesis. In some embodiments, DNA sequencingdata on TCR or antibody repertoires are obtained using methods known inthe art, and then synthetic DNA libraries are engineered from sequencesidentified through bulk sequencing. In some embodiments, the synthesizedDNA libraries comprise TCRs or antibodies known to bind to antigens ofinterest through methods comprising yeast display, mammalian display, ormammalian cell activation assays. DNA oligonucleotides can be designedsuch that they comprise libraries of overlapping, complementarysequences that hybridize when incubated together. Libraries of hundreds,thousands, tens of thousands, or hundreds of thousands of syntheticoligonucleotides can be manufactured by microfluidic or array-basedmethods, for example, by commercial providers such as Twist Bioscience,Agilent Technologies, or LC Biosciences. The libraries ofoligonucleotides can be then assembled into DNA sequences of hundreds orthousands of nucleotides, using 5′ exonuclease, DNA polymerase, and DNAligase (e.g., “Gibson Assembly”, Gibson et al. Nat Methods. 2009 May;6(5):343-5). For example, T5 exonuclease, Taq polymerase, and Taq ligaseare mixed in a reaction comprising overlapping oligonucleotides,nucleotides, DTT, MgCl₂, and buffer, and then incubated at 50° C. for 60minutes. In some embodiments, Gibson Assembly is used to synthesizecircular clones, for example, plasmid expression constructs. If thesynthetic DNA is circular, the DNA can be transformed into bacteria toproduce nanogram or more quantities of plasmid. Another method thatgenerates linear synthetic DNA comprises mixing overlappingoligonucleotides and performing PCR using a thermostable polymerase. Inorder to make circular DNA, these linear PCR products can be thensubcloned into plasmid expression constructs using methods comprisingrestriction enzymes and DNA ligase, Gibson Assembly, or blunt endcloning. Any of these DNA synthesis methods can be parallelized through96-well plate, 384-well plate, microfluidic, or robotic processingsystems.

DNA libraries of antibodies, TCRs, or any other target gene can be alsogenerated through isolation and lysis of single cells, followed bynucleic acid amplification. Single B cells can be isolated into 96-wellplates, and then heavy and light chain immunoglobulin transcripts can belinked using a method known in the art, for example, multiplexed“overlap extension” RT-PCR (Oleksiewicz EP 1921144 B1). In overlapextension RT-PCR, or OE-RT-PCR, for immunoglobulin amplification fromsingle cells, a pool of primers can be designed that bind and amplifyall possible heavy chain genes and all possible light chain genes. Theheavy chain primers can also comprise subsequences with complementarityto the light chain primers. During OE-RT-PCR, the complementarysubsequences can hybridize and a polymerase can generate a fusedpolynucleic acid from hybridized single stranded heavy and light chainimmunoglobulin. In this fashion, the single cell context of the heavyand light chain immunoglobulin can be maintained.

DNA libraries of antibodies, TCRs, or any other target gene can begenerated by other methods, for example, those involving OE-RT-PCR andmicrofluidics from populations of more than ten thousand cells. Oneexemplary method disclosed in Johnson EP2652155, which is incorporatedby reference in its entirety herein, involves use of a dropletmicrofluidic device. The droplet microfluidic device inputs anoil/surfactant mix, lysis and RNA capture mix, and a cell suspension andoutputs single-cell emulsions into standard thermocycling microtubes.The oil/surfactant mix is based on mineral oil or fluorocarbon oil. Thelysis and RNA capture mixture contains oligo-dT coated magnetic 1 μmbeads that capture messenger RNA (mRNA) transcripts from the singlecells. The cell encapsulation device is comprised of three pressurepumps, a microfluidic droplet chip, and imaging apparatus. Themicrofluidic chip is fabricated from glass and channels are etched to 50μm×150 μm for most of the chip's length, and narrow to 55 μm at thedroplet junction. Droplet size depends on pressure, but typicallydroplets of ˜40 μm are optimally stable and appropriately sized for thesingle cell emulsions. Droplet generation rates also depend on pressure,but are typically up to 3 kHz and capture 3 million cells per hour. Celllysis methods comprise surfactant based methods, for example TritonX-100, NP-40, Tween 20, Tween 80, or SDS. The emulsions are incubated at50° C. for 30 minutes, and then the beads are extracted from theemulsion using a solvent such as ethyl acetate. Next, the mRNA-boundbeads are injected back into emulsions for OE-RT-PCR, using microfluidicchips similar to the cell encapsulation chips described above. Forexample, to generate TCRαβ libraries, independent TCRα and TCRβ minoramplicons are generated in multiplex; these are then fused to generate asingle major amplicon comprised of both TCRα and TCRβ. The TCRαβ primerpool includes a universal primer for β constant (Cβ) and α constant (Cα)regions. This abrogates the need for a large pool of J region primers.Additionally, the C region primers are designed to either capture theendogenous C region genotypes or isotypes, or the primers are designedto ignore endogenous C region genotypes or isotypes. The TCRαβ primerpool also includes 43 primers that bind to all possible V segments forTCRαβ and TCRβ. Thus, the primers amplify across the full variableregion of each monomer to produce 450 bp minor amplicons. Exemplaryprimers for TCRβ V gene is provided herein as SEQ ID NO: 17-19, anexemplary primer for TCRβ C gene is provided herein as SEQ ID NO: 20,exemplary primers for TCRα V gene are provided as SEQ ID NO: 21-23 andan exemplar primer for TCRα C gene is provided herein as SED ID NO: 24.The bead emulsions are then subjected to OE-RT-PCR using a reactioncomprising an RT, gene-specific primers, a thermostable polymerase suchas Taq, and thermocycling consisting of reverse transcription (42° C.for 60 minutes), denaturation (95° C. for 30 seconds), 30 cycles ofamplification (95° C. for 15 seconds, 62° C. for 60 seconds, and 68° C.for 3 minutes), followed by a final extension at 68° C. for 5 minutes.Because a plurality of droplets contains only a single mRNA-bound bead,the native TCRαβ pairing of the input T cell is maintained in the TCRαβlinkage library. Similar methods can be used to generate linked heavyand light chain immunoglobulin DNA libraries. For example,immunoglobulin primer sets comprising a polynucleotide of any of SEQ IDNO: 1-8 can be used. SEQ ID NO: 1-3 provide exemplary primer sequencesfor IGG V gene, SEQ ID NO: 4 provides an exemplary primer sequence forIGG C gene, SEQ ID NO: 5-7 provide exemplary primer sequences for IGK Vgene, and SEQ ID NO: 8 provides an exemplary primer sequence for IGK Cgene. Primers for the immunoglobulin C regions are eitherisotype-specific, genotype specific, or are universal primers designedamplify any C region sequence. In some embodiments, the TCR orimmunoglobulin subunits are linked with a polynucleic acid sequenceencoding a porcine teschovirus-1 (P2A) amino acid sequence. In someembodiments, the TCR or immunoglobulin subunits are linked with apolynucleic acid sequence encoding a Gly-Ser peptide linker. In someembodiments, the TCR or immunoglobulin subunits are linked with apolynucleic acid sequence encoding an Internal Ribosome Entry Site(IRES). In other embodiments, the TCR or immunoglobulin subunits arelinked with artificial linker sequences with no significant homology toany known endogenous sequences.

The DNA libraries of linked TCRαβ or heavy and light chainimmunoglobulin can be converted to recombinant expression constructsusing methods known in the art, for example, the method described inJohnson U.S. Pat. No. 9,422,547 B1, which is incorporated by referencein its entirety herein. The exemplary method described in Johnson U.S.Pat. No. 9,422,547 B1 uses nested outer PCR primers to add adapters withoverhangs for Gibson Assembly to the 5′ and 3′ ends of the ampliconlibrary. Primers for the C regions can be either isotype-specific,genotype specific, or are primers designed amplify any C regionsequence. T5 exonuclease and Taq ligase are mixed in a reactioncomprising the TCRαβ or immunoglobulin insert, a linearized plasmidbackbone with subsequences complementary with the insert, DTT, MgCl₂,and buffer, and then incubated at 50° C. for 60 minutes. The plasmidbackbone comprises a promoter, a poly(A) signal sequence, and C regionsequence not amplified through OE-RT-PCR. The C region matches theisotype or genotype of the linked amplicon, or is designed to fuse theamplicon with a non-native isotype or genotype. The library is thentransformed into E. coli and spread on LB-ampicillin plates. The plasmidlibrary is then purified with a Maxi prep kit. The purified Maxi preplibrary contains tens of thousands, hundreds of thousands, or millionsof clones. Some workflows require a second round of Gibson Assembly. Forexample, if the one or both of the full C regions are not amplified inthe original OE-RT-PCR, it may be necessary to clone a C region betweenthe TCRαβ or heavy and light chain immunoglobulin. In some embodiments,a promoter, P2A, or IRES sequence is cloned at the same time. Theinserted sequences are synthesized by assembling a pool ofoligonucleotides using Gibson Assembly or PCR, and then Gibson Assemblycan be used to insert the polynucleic acid insert into the plasmidlibrary. This reaction can be performed on tens of thousands, hundredsof thousands, or millions of clones in parallel. The final result is alibrary of tens of thousands, hundreds of thousands, or millions ofTCRαβ or heavy and light chain immunoglobulin clones that express fullyfunctional proteins, which retain the native pairing of the originalsingle cell inputs.

In some embodiments, single cell amplification methods are used togenerate single cell cDNA libraries for any transcript, set oftranscripts, or full single cell transcriptomes using various methodsfor nucleic acid barcoding, for example, as described in Johnson U.S.Ser. No. 15/159,674, which is incorporated by reference in its entiretyherein.

The exemplary method disclosed in Johnson U.S. Ser. No. 15/159,674comprises delivering a clonal polynucleic acid barcode with a singlecell into a reaction vessel, microfluidic chamber, or an emulsionmicrodroplet. One method is to affix polynucleic acids that comprisebarcodes to solid supports comprising spherical beads with 1 μm, 5 μm,or 10 μm diameter, made of magnetic material to facilitate nucleic acidpurification. Oligonucleotides are modified with NH2 and affixed toepoxy silane or isothiocyanate coated glass beads, or oligonucleotidesare disulfide modified and attached to mercaptosilanized glass supports.For droplet encapsulation, bead solutions are mixed with cells, and thendiluted such that a plurality droplets contain a single cell and asingle bead. Because such methods result in a plurality of emptydroplets or droplets with only a single bead or only a single cell, insome methods, cells and beads are first encapsulated into droplets inseparate streams or separate devices, and then the cell- andbead-containing droplets are fused to generate a plurality of dropletsthat contain a single cell and a single bead. Depending on theapplication, a plurality of single cells can be encapsulated withmultiple barcoded beads. Such methods enable trace back of individualbarcodes to single cells, even if there are multiple barcodes for aplurality of single cells. Other methods comprise biotin-streptavidinand covalent conjugation chemistries. Another method is to affixpolynucleic acids that comprise barcodes to antibodies, which are boundto cells prior to delivering the cells to reaction vessels, microfluidicchambers, or emulsion microdroplets. Methods for conjugating antibodiesto nucleic acids available in the art can be employed, for example,biotin-streptavidin or covalent conjugation chemistries. Cell lysismethods can comprise surfactant based methods, for example Triton X-100,NP-40, Tween 20, Tween 80, or SDS. In some embodiments, the emulsionsare incubated at 50° C. for 30 minutes, and then the beads are extractedfrom the emulsion using a solvent such as ethyl acetate. Next, theRNA-bound beads are recovered from the emulsion and then amplified indroplets or reaction vessels using the methods described above, withsome modifications specific to nucleic acid barcoding. In nucleic acidbarcoding, the first strand cDNA can be labeled with the nucleic acidbarcode fused to the transcript-specific first strand primer. Universalprimers 5′ to the nucleic acid barcode can be used in PCR to amplify aplurality of barcoded RT-PCR amplicons. Alternatively, RNA can be primedand amplified separately from the barcode sequence, and then the barcodeand cDNA amplicons can be fused in an overlap extension PCR inside ofemulsion microdroplets. Alternatively, first strand cDNA barcoding canbe effected with RT in the lysis mixture, without the requirement toinject RNA-bound beads for an RT-PCR amplification. In these methods,the cDNA-bound beads can be extracted from the emulsion and the barcodedcDNA can be subjected to “bulk” PCR, i.e., PCR without an emulsion. Thefinal result of any of these methods can be a library of tens ofthousands, hundreds of thousands, or millions of barcoded cDNA clonesthat express fully functional proteins, which enable trace back of cDNAswith the same barcode back to a single originating cell. The cDNAs arenot necessarily full length, for example, peptide:MHC complexes do notrequire full cDNA for functional analysis. In some embodiments, a targetlibrary comprises NY-ESO-1 target sequence (SEQ ID NO: 13), or MART-1target sequence (SEQ ID NO: 16), engineered into two different mammalianclones.

Once reformatted as circular plasmids, libraries of cDNAs can beintroduced into mammalian cells for protein production. For example,TCRαβ expression constructs can be packaged into lentivirus or any othervector known in the art and then used to transduce the Jurkat J.RT3-T3.5cell line (ATCC) or other cells, which lack TCRαβ expression and thushave no cell surface TCR. In one specific embodiment, first, startingwith the TCRαβ plasmids Vesicular Stomatitis Virus G (VSV-G) pseudotypedlentiviral particles are generated using the 3rd generation ViraSafeLentiviral Packaging System (Cell Biolabs) and Lenti-Pac 293Ta cells(GeneCopoeia). Lentiviral copy number can be determined using theLenti-X qRT-PCR Titration Kit (Clontech) to normalize transduction. Inthe exemplary embodiment, 10⁵ or 10⁶ J.RT3-T3.5 cells are transducedwith a library of lentiviral construct and then selected with Puromycinfor 14 days. In the exemplary embodiment, FACS analysis demonstrates15-30% transduction efficiency. In other specific embodiment, CHO Flp-In(provided commercially by Life Technologies) cells are transfected fortargeted genome integration of heavy and light chain immunoglobulinlibraries. Whereas lentivirus integrate randomly into a mammaliangenome, plasmids engineered for Flp-In will only integrate at an FRTsite in a cell's genome. CHO Flp-In cells have been previouslyengineered to contain an FRT site at a single location in the genome. Toengineer a library of antibody-expressing cells, a ratio of 2:1 Flprecombinase vector to antibody plasmid library is used to electroporatefour million CHO Flp-In cells in Ingenio buffer (Mirus Bio). After twodays in growth medium without selection, the growth medium issupplemented with 600 g/mL hygromycin, which selects against cellslacking stable integrants. After three weeks, colonies are counted, suchthat in a successful experiment, approximately ˜1% of the electroporatedcells result in stable integrants. CHO Flp-In cells are engineered withsecreted or membrane-bound antibodies, depending on the requirements ofdownstream experiments. Other methods known in the art can be used toengineer protein expression constructs into the genomes of mammaliancells, for example, random integration of retroviruses, CRISPR/Cas9,Transcription Activator-Like Effector Nucleases (TALENs), and zincfinger nucleases. Any of the methods can be employed to obtain a libraryof cell clones that express thousands, tens of thousands, hundreds ofthousands, millions, or hundreds of millions of different transcript andprotein sequences of interest. In some embodiments, an example targetlibrary comprises NY-ESO-1 target sequence (SEQ ID NO: 13), or MART-1target sequence (SEQ ID NO: 16), engineered into two different mammalianclones.

2) Preparation of Target Cells, Intermediary Cells, and Inducer Cellsfor Functional Assays

Some aspects of the present invention relate to a method of partitioningsingle clonal cells with their target cells, or single clonal cells withintermediary cells and target cells. To facilitate high-throughputanalysis, partitioning of cells can be achieved by encapsulation intoaqueous-in-oil droplets using droplet microfluidic chips. Anymicrofluidic chips known in the art can be employed. For example,microfluidic chips that can be used for various embodiments of thepresent invention include, but not limited to those fabricated fromglass, plastic, PDMS, or other polymers. One specific embodiment employsa microfluidic chip fabricated from glass, with channels etched to 50μm×150 μm for most of the chip's length, and which narrow to 55 μm atthe droplet generation junction. Fluid is pumped through themicrofluidic chips using pressure pumps or syringe pumps. Cells areinjected into droplets in two streams. For example, APCs are injected inone stream and TCR-expressing cells are injected in a second stream.Typically, TCR-expressing cells are injected at 10,000-20,000 cells permicroliter, and APCs are injected at a slightly lower concentration, forexample, 2,000-5,000 cells per microliter, such that most droplets thatcontain an APC contain only a single APC. The droplets containing thecell mixtures are in the range of 20-200 μm. The ratio of inducer totarget cells varies from application to application, but it is desirablefor the partitions to contain single inducer cells, enabling detectionof functional interaction between a clonal cell and its target. In someembodiments, cells are encapsulated into gels rather than aqueoussolutions. For example, agarose gels are used to embed and encapsulatecells of interest. Reaction vessels such as 96-well plates, 384-wellplates, or microfluidic chamber chips can be used if the size of theclone library does not exceed 10,000 genetically distinct clones. Flowcytometry or manual pipetting can be used to distribute cells into96-well plates. Cells can be distributed into microfluidic vessel chips(for example, from vendors such as Fluidigm) using pressure pumps orsyringe pumps, and microfluidic microwell valves are used to capturecells into microfluidic chambers. Regardless of whether droplets orreaction vessels are used to partition mixtures of cells, the mixturesof cells can be incubated in a way that enables the inducer cells toinduce transcriptional changes in the target cells and/or intermediarycells, for example, RPMI, DMEM, or IMDM, supplemented with 10% fetalcalf serum (FCS), at 37° C. in a tissue culture incubator.

In some embodiments, a glass microfluidic chip is used to inject CHOcells into 35 μm radius droplets in RPMI with 10% FCS, with the oilphase comprising fluorocarbon oil and surfactant. Sytox Orange andCalcein-AM (ThermoFisher) are included in the media to stain for deadand live cells, respectively. We then overlay the emulsions with a layerof mineral oil to prevent fluorocarbon oil evaporation but enable gasexchange. The emulsions are then incubated in a microcentrifuge tube ina conventional tissue culture incubator at 37° C., 5% CO₂. We then useour fluorescent microscope to assess live/dead staining. In a typicalexperiment, 49/50 cells are still alive after 16 hours, and 45/50 cellsare still alive after 24 hours. After 72 hours, >85% of cells are stillintact, but no longer fluoresce sufficiently for live/deaddetermination.

Target and inducer cells incubated in emulsion microdroplets can belysed to generate a plurality of polynucleic acids that fuse clonalsequences from the inducer cell with induced transcripts from the targetcell. Such protocol can retain proper pairing between inducer clones andtarget cells. Cell culture media which is optimal for functional studiesis not necessarily optimal for cell lysis and enzymatic polynucleic acidamplification. To address this issue, a droplet microfluidic chip designthat fuses cell-containing droplets with lysis/bead mix can be used.

In some embodiments, droplet fusion is driven by interfacial forceswhere two droplets have a larger interfacial area than a single dropletof the same volume. To achieve this situation, the continuous phaseseparating the two droplets can be removed. For example, when the twodroplets have close contact with each other, a thin liquid bridge formsbetween the two droplets due to molecular attractions between thedroplets. The curvature meniscus formed around the bridge creates animbalance of surface tension which quickly merges the two droplets.Fusion of emulsion microdroplets is either passive (i.e., not requiringoutside energy) or active (i.e., requiring outside energy) (assummarized, for example, in Xu Micro and Nanosystems 2011 3:131-136).Passive methods can rely on the structure of the microchannel or surfaceproperties of the microchannel. On the other hand, active dropletcoalescence can use energy supplied by an outside source, for example,by applying a magnetic, electric, or temperature field.

In one exemplary embodiment, one chip design, manufactured in PDMS,comprises two aqueous input channels and two oil input channels. Theaqueous/oil inputs are in two pairs, i.e., one aqueous inlet is pairedwith one oil inlet. One aqueous/oil inlet pair is approximately 100 μmin width or diameter, and the other is approximately 50 μm in width ordiameter. Mixtures of cells in ˜40 μm emulsion microdroplets areinjected into the 50 μm channel using a pressure pump set atapproximately 100 mbar. A mixture of oligo-dT magnetic beads andTween-20 surfactant in an aqueous binding buffer, in ˜80 μm droplets, isinjected into the 100 μm channel using a pressure pump set atapproximately 100 mbar. The droplets streams merge into a single channelsuch that they co-flow at periodicities controlled by the pressure orflow rate of the inlet lines. The two oil inlet lines are used toachieve droplet periodicity such that each cell mixture droplet ispaired with a single lysis and bead mix droplet. Using a power supply(Mastech) and an inverter (TDK), a 7 V AC electrical current is appliedto a 160 μm stretch of widened droplet co-flow channel. The current isapplied by injecting a 1M NaCl solution into a channel unconnected tothe droplet co-flow channel, but close enough that the AC current isconducted into 160 μm stretch of widened co-flow channel. The ˜80 μmlysis/bead mix droplet slows down slightly and deforms in the widenedchannel. The ˜40 μm cell mixture droplets do not slow down in thewidened channel, ensuring that each cell droplet is in contact with alysis/bead droplet. Simultaneous application of electric current resultsin fused, diluted droplets, which are then incubated off-chip to bindpoly(A) RNA to the oligo-dT beads. A typical experiment in the settingachieves >98% droplet fusion at a throughput of ˜500 droplets persecond, with 100% cell lysis.

The interaction between a TCR and its cognate peptide:MHC target caninduce transcriptional responses in both the TCR-expressing cell (e.g.,primary T cell or TCR-engineered Jurkat cell) and thepeptide:MHC-expressing cell (e.g., primary APCs or engineered APCs).Depending on which functional cellular interaction is of interest,primer sets can be designed to link peptide:MHC sequence with T celltranscriptional response, or TCR sequence with APC transcriptionalresponse. In some embodiments, it is desirable to investigate theinteraction comprehensively, e.g., link peptide:MHC, TCR, T cellresponse, and APC response. To link peptide:MHC sequence with T celltranscriptional response, APCs can be incubated with TCR-expressingcells in emulsion microdroplets in a combinatorial screen, using thepartitioning methods described above. The recombinant APCs can beengineered to express a library of peptide:MHC targets, with a specificbarcode indicating each peptide:MHC target in the library. Afterincubation for 6, 12, 18, 24, 36 hours, or more in emulsionmicrodroplets, the cell mixture emulsion microdroplets can be fused withlysis/bead emulsion microdroplets using the methods described above. TheRNA-bound beads can be then injected into emulsion microdroplets formultiplex OE-RT-PCR. Primers can be introduced into the emulsionmicrodroplets that amplify at least one T cell activation marker, forexample, Interferon Gamma (IFNg), CD69, or Interleukin-2 (IL-2). Theprimers can be designed to span across introns, such that noamplification from background genomic DNA takes place, and the ampliconsare 100 bp-300 bp in size. In some embodiments, one primer of each Tcell activation primer pair has a polynucleic acid subsequence withcomplementarity to one primer of the barcode amplification primer pair.The complementary subsequences hybridize during OE-RT-PCR, so that aplurality of linked amplicons is generated. In this way, peptide:MHCtarget sequences are linked to functional responses in T cells. In anexemplary embodiment, a target library comprises clones engineered witha polynucleotide of NY-ESO-1 target sequence (SEQ ID NO: 13) and apolynucleotide of MART-1 target sequence (SEQ ID NO: 16), and a primerset that comprises target barcode primers (e.g., SEQ ID NO 14-15),primers for IFNG (SEQ ID NO: 25-26), and primers for IL-2 (SEQ ID NO:27-28). Sequencing adapters (e.g., Illumina sequencing adapters) can beadded to the library of linked amplicons using nested, tailed-end PCR,as described above. The peptide:MHC and T cell activation markerpairings can be identified and quantified by deep sequencing the linkedamplicons, for example, obtaining 100,000, one million, or ten millionsequences from the library of linked peptide:MHC and T cell activationmarker complexes. Bioinformatics can be then used to match the sequencedbarcodes with peptide:MHC by searching a database of peptide:MHCbarcodes, which was generated using any of the methods above. In someembodiments, it is beneficial to in parallel generate hybridizedamplicons that link TCR sequences to peptide:MHC sequences, and TCRsequences with T cell activation markers. For such embodiments, theOE-RT-PCR amplification mixtures can also include primers that link TCRβpolynucleic acids with peptide:MHC barcodes and/or T cell activationmarkers. The TCR primer set can amplify from the most 5′ end of the TCRβV region across to a universal primer that sits in the Cβ region. The Cβprimer can have a polynucleic acid subsequence with complementarity toone primer from the barcode amplification primer pair, and to one primerfrom each of the T cell activation marker primer pairs. The TCRβamplicons can be ˜400-500 bp in size. The primer set that includesprimers for peptide:MHC barcodes, T cell activation markers, and TCRβcan generate the following amplicons: peptide:MHC linked to T cellactivation markers, TCRβ linked to peptide:MHC, and/or TCRβ linked to Tcell activation markers. Sequencing adapters (e.g., Illumina adapters)can be added to these amplicons using nested, tailed-end PCR, asdescribed above. The library (e.g., Illumina library) can be then deepsequenced to obtain 100,000, one million, or ten million sequences.Bioinformatics can be then used to process the raw sequences, and thenmatch peptide:MHC to TCRβ, TCRβ to T cell activation markers, and/orpeptide:MHC to T cell activation markers. In this way, the combinatorialscreen yields a list of cognate pairs of peptide:MHC and TCRs that bindand activate cellular phenotypes of interest. An even more comprehensivemixture can also generate TCRαβ linkage amplicons, such that theinteractions between APCs and T cells can be used to identify linkedTCRαβ of interest, which are then expressed as full length recombinantTCRαβ, and further analyzed for in vitro and in vivo function.

Other primer mixes can be used if other T cell functional responses areof interest. For example, so-called immune “checkpoint” genes act asco-stimulatory or co-inhibitory regulators of T cell activity.Checkpoint molecules are typically expressed on the surface of T cellsor T cell target cells, and interact with other co-stimulatory orco-inhibitory molecules on the surface of the same cell or another cell.Checkpoint molecules and the utility of “checkpoint inhibition” incancer therapy are known in the art (e.g., Shin Current Opinion inImmunology 2015, 33:23-35). These networks of co-stimulatory orco-inhibitory molecules are activated or antagonized by a variety ofmolecules, including monoclonal antibodies, and such modulatorymolecules effect changes in T cell phenotype. Combinatorial screens canbe performed on various combinations of activating or antagonizingmolecules, or molecules with unknown function, to induce transcriptionalchanges in target T cells. This can be achieved, for example, bypartitioning a library of antibody-secreting CHO cells (inducer cells)with checkpoint-expressing cells (target cells, e.g., T cells). In someembodiments, the checkpoint-expressing cells are non-engineered primaryT cells, or primary T cells transduced to express a checkpoint receptorprotein. The antibody-secreting CHO cells can comprise a library ofantibodies with known activities against checkpoint molecules, or alibrary of antibodies with unknown function, for example, a librarygenerated from antibody-expressing cells isolated from a mouse immunizedwith a checkpoint protein. In any scenario, antibody-expressing cellscan be isolated into emulsion microdroplet partitions withcheckpoint-expressing target cells. Ratios of antibody-expressing cellsto target cells in this setting can be 1:1, 1:2, or 1:5, or any ratio inbetween if the functions of the antibodies are unknown. Optimal ratiosof antibody-expressing cells to target cells in this setting are 1:1,2:1, or 5:1 if the goal is to identify combinations of antibodies thatinduce expression of checkpoint molecules. After incubation for 6, 12,18, 24, 36, or more hours in emulsion microdroplets, the cell mixtureemulsion microdroplets can be fused with lysis/bead emulsionmicrodroplets using the methods described above. The RNA-bound beads canbe then injected into emulsion microdroplets for multiplex OE-RT-PCR. Inthis application, primers for OE-RT-PCR can comprise antibody-specificprimers and checkpoint-molecule specific primers. The antibody primerpool can include a universal primer for the heavy chain constant (C)region. This abrogates the need for a large pool of J region primers.The primer pool can also include primers that bind to all possible Vsegments for IgG. The primers can amplify across the full variableregion of each Ig monomer, i.e., FR1, CDR1, FR2, CDR2, FR3, CDR3, andFR4 for heavy and light chain Igs. Antibody heavy chain amplicons can be400-450 bp. At least one checkpoint transcript primer pair can beincluded, for example, a primer pair for LAG-3, PD-1, TIM-3, CEACAM-1,CD200R, CTLA-4, TIGIT, or BTLA. General proliferation or activationmarkers can also be included, such as IFNg or IL-2. Some primer poolsinclude primers for all of these transcripts, or subsets of the list.The primer pool can also comprise the full transcriptome of the T cells.The primers can be designed to span across introns, such that backgroundgenomic DNA does not contaminate the amplification signal. Amplicons forthese transcripts can be between 100-300 bp, 200-500 bp, 300-600 bp orless than 1000 bp. The antibody C region primer can comprise asubsequence with reverse complementarity with a subsequence of onemember of the primer pair for each of the checkpoint transcripts. Thecomplementary polynucleic acid subsequences enable OE-RT-PCR to generatemajor amplicons that link an antibody sequence from a CHO cell withcheckpoint sequences from a target cell. Sequencing adapters (e.g.,Illumina sequencing adapters) can be added to the library of linkedamplicons using nested, tailed-end PCR, as described above. The antibodyand T cell checkpoint marker pairings can be identified and quantifiedby deep sequencing the linked amplicons, for example, obtaining 100,000,one million, or ten million sequences from the library of linkedcomplexes. Bioinformatics can be then used to quantify the checkpointtranscripts linked to each antibody of interest. In some embodiments, itis beneficial to also identify clonality of the reactive T cell clone.For example, if multiple antibody-expressing CHO cells are isolated intoemulsion microdroplets with target cells, functional combinations ofantibodies can be of interest. In this situation, T cell clones can beidentified by including TCRβ primers in the OE-RT-PCR mix. In someembodiments, the T cells can be engineered to express transcripts withbarcodes, such that the barcodes are used to identify the T cell clonesthat are reactive to antibody combinations. In any experimental design,the bulk sequencing data can have utility for identification offunctional relationships among co-stimulatory and co-inhibitorycheckpoint molecules. For example, activation of OX40 can result indown-regulation of PD-1 or CTLA4, inhibition of PD-1 can result inactivation of OX40, and so on. In another example, a mixture of twoantibodies activates T cells more effectively than any other mixtures,as evidenced by a large plurality of bulk sequencing data that link theantibody sequences with IFNg and IL-2 proliferation and activationmarkers. In some embodiments of the invention, transcripts of interestare either up-regulated or down-regulated.

In some embodiments, a primer set that links NK cell activity(intermediary cells) with antibody-expressing cells (inducer cells) canbe used. For example, a population of CHO cells is engineered to expressa library of secreted antibodies. Another population of CHO cells isengineered to express antigens of interest. Alternatively, tumor cellsare used as antigen-expressing cells. In a typical combinatorial screen,a plurality of single cells from a library of tens, hundreds, thousands,hundreds of thousands, or millions of antibody-expressing CHO clones arepartitioned with antigen-expressing cells. If the antigen-expressingcells comprise a diverse population of clones, the ratio ofantibody-expressing cells to antigen expressing cells can be 1:2, 1:1,2:1, or any ratio in between. If the antigen-expressing cells comprisecancer cells, the ratio of antibody-expressing cells to cancer cells canbe 1:1, 1:5, 1:10, 1:100, or any ratio in between. The mixtures ofantibody-expressing cells and antigen-expressing cells can bepartitioned into emulsion microdroplets with NK cells. We refer to theNK cells as intermediary cells because the the antibody-expressing cellsinduce changes in NK cell expression via binding of secreted antibody tothe target cells, instead of through direct cell-to-cell interactionsbetween the antibody-expressing cells and the target cells. Afterincubation for 6, 12, 18, 24, 36, or more hours in emulsionmicrodroplets, the cell mixture emulsion microdroplets can be fused withlysis/bead emulsion microdroplets using the methods described above. TheRNA-bound beads can be then injected into emulsion microdroplets formultiplex OE-RT-PCR. In this application, primers for OE-RT-PCR cancomprise antibody-specific primers and NK activation primers.Antibody-specific OE-RT-PCR primers are described above. NK transcriptsthat are up-regulated upon activation can include effectors (IFNg;TNFa), proteases (Granzyme A [Gzma]; Granzyme B [Gzmb]), transcriptionfactors (T Box Transcription Factor 21 [Tbx21/T-bet]; Eomesodermin[Eomes]; PU Box Transcription Factor [PU.1]; Inhibitor of DNA Binding 2[Id2]), and signaling adaptor proteins (DAP12; Spleen AssociatedTyrosine Kinase [Syk]; Zeta-Chain-Associated Protein Kinase 70 [Zap70]).Transcripts of interest can also comprise targets that aredown-regulated on NK cell activation. The NK cell activation primer setcan comprise at least one NK cell activation transcript target, forexample, they can comprise two, five, ten, 100, or 1,000 targets, or thefull transcriptome of NK cells. The primers can be designed to spanacross introns, such that background genomic DNA does not contaminatethe amplification signal. Amplicons for the NK activation transcriptscan be 100-300 bp, 200-500 bp or less than 1000 bp. The antibody Cregion primer can comprise a subsequence with reverse complementaritywith a subsequence of one member of the primer pair for each of the NKcell activation transcripts. The complementary polynucleic acidsubsequences enable OE-RT-PCR to generate major amplicons that link anantibody sequence from a CHO cell with NK cell activation sequences. Insome embodiments, an example primer set comprises primers for IGG V gene(e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4), primersfor GZMB (e.g., SEQ ID NO:9 and SEQ ID NO:10), and primers for TBX21(e.g., SEQ ID NO:11 and SEQ ID NO:12). Sequencing adapters (e.g.,Illumina sequencing adapters) can be added to the library of linkedamplicons using nested, tailed-end PCR, as described above. The antibodyand NK cell activation marker pairings can be identified and quantifiedby deep sequencing the linked amplicons, for example, by obtaining100,000, one million, or ten million sequences from the library oflinked complexes. Bioinformatics can be then used to quantify the NKactivation transcripts linked to each antibody of interest. In this way,antibodies that induce NK cells can be identified through a functionalassay that involves three cell types: NK cells (intermediary cells),antigen-expressing cells (target cells), and antibody-expressing cells(inducer cells).

In some embodiments, the transcripts induced in the target cells areuncharacterized, or the transcriptional signature of the target responseis complex, requiring quantification of hundreds or thousands oftranscripts. In those cases, methods that quantify the fulltranscriptome of gene targets can be used. For example, uniquepolynucleic barcodes are affixed to solid supports, such as beads, usingthe methods described above, and are delivered to emulsion microdropletswith cell mixtures. Barcoded polynucleic acids from the beads, alsocomprising oligo-dT subsequences, can be used to barcode the fulltranscriptome of a target cell. This can be achieved through OE-RT-PCRor through first strand labeling. Then, OE-RT-PCR or OE-PCR can be usedto generate major amplicons comprising polynucleic acid sequencesindicative of the inducer clone. For example, peptide:WIC can be linkedto the full transcriptome of a TCR-expressing cell, or an antibodysequence from an antibody-expressing cell can be linked the fulltranscriptome of a T cell. Such methods are also possible where theinducer clone does not directly interact with the target cells, forexample, NK cells activated through antibodies binding to tumor cells,as described above. Nested, tailed-end PCR can be used to attachsequencing adapters (e.g., Illumina sequencing adapters) to a pluralityof the major amplicons. Then, bulk sequencing can be performed to obtainhundreds of thousands, millions, hundreds of millions, or billions ofsequences. Bioinformatic algorithms can be used to identify transcriptsin target cells or intermediary cells that are up- or down-regulated inresponse to inducer cells. Such methods can be used to discover novelbiomarkers for functional cellular interactions.

The methods described above are provided as examples, and any variantsthereof can be adopted to achieve similar utility. For example, nucleicacid amplification can be effected through padlock probes or ligasechain reaction. Though most of the protocols described above use RNAsequences for clonal identification, it is also possible to use genomicDNA sequences for clonal identification. For example, a library ofinducer clones can be made by directed CRISPR/Cas9 genome editing, orrandom insertion of a polynucleic acid of interest into a library ofinducer clones. In such situations, the genomic DNA sequence of interestcan be amplified and linked to transcripts in the target cells. In someapplications, changes other than transcriptional changes can be inducedin the target cells. For example, inducer cells can induce epigeneticchanges in the target cell's genome. In some applications, inducer cellscan change protein profiles of target cells. Such changes can bequantified by binding nucleic-acid barcoded antibodies to the targetcells, such that the barcoded antibodies can be amplified and linked topolynucleic acid sequences for clonal identification in the inducercells.

In some embodiments of the invention, a polynucleic acid barcode isdelivered to a droplet or vessel that contains a mixture comprisingtarget and inducer cells. This polynucleic acid barcode can be affixedto a solid support, such as a bead, antibody, or cell. The cells can belysed and RNA from the mixture of cells is fused with polynucleic acidbarcode. Transcript cDNAs from target and inducer cells can be thensequenced and traced back to the droplet or vessel using the polynucleicacid barcodes. Thus, in some embodiments, transcript cDNAs from targetand inducer cells are never directly fused, but rather the combinationsare linked bioinformatically through the polynucleic acid barcodes.

In some embodiments of the invention, cells, cell mixtures, or emulsionmicrodroplets are labeled with RFIDs, electronically indexed solidsupports, light-triggered microtransponders (e.g., Mandecki US20160175801), quantum dots, colorimetric indexes, fluorescent markers,or other identifying “barcodes” that are not based on polynucleic acids.These identifiers can be used to identify clones, memorialize laboratoryprotocols used to process mixtures of cells, or indicate the result of abiological assay. Such identifying barcodes can be affixed to orcomprise solid supports, such as microchips or beads of less than 50microns at the widest dimension, affixed to proteins, or engineered intocells as expression constructs responsive to a stimulus. In someembodiments, one population of TCR-expressing clones, for example, CD4+T cells, is labeled with the same RFID barcode. A second population ofTCR-expressing clones, for example, CD8+ T cells, is labeled with asecond RFID barcode. Then, these two populations of cells are mixed. Insome embodiments of the invention, the population of the RFID-taggedTCR-expressing clones are encapsulated into emulsion microdroplets witha library of peptide:MHC-expressing cells, as described above. The RFIDtags can be then used to sort microdroplets into CD4+ and CD8+emulsions. In this way, the RFID barcode enables further de-multiplexingbeyond a nucleic acid barcode or TCR clone. Two, ten, 100, 1,000,100,000, or millions of different RFID particles can be used. In someembodiments, the identifying index is a fluorescent marker, andcell-containing droplets are sorted with flow cytometry, or FACS. Insome embodiments, a biological assay taking place inside emulsionmicrodroplets results in production of a fluorescent marker, and thencell-containing droplets are sorted with flow cytometry. In someembodiments, a single fluorescent wavelength is used, andcell-containing droplets are sorted as positive or negative based on afluorescence threshold that indicates a positive readout in thebiological assay. Polynucleic acid barcodes can also be affixed toparticles with RFIDs, for example, to link RFID with deep sequencingdata. The particles with RFIDs can also be soaked in drugs, or coatedwith antibodies or proteins, which can then be used in functional assaysand de-multiplexed with an RFID reader. In some embodiments, the RFID,electronically indexed solid supports, quantum dots, colorimetricindexes, fluorescent markers, or other identifying “barcodes” that arenot based on polynucleic acids are used to trace an incubation protocol.For example, there is an interest in incubating TCR-expressing cellswith peptide:MHC-expressing cells for 2 hr, 6 hr, 10 hr or more.RFID-tagged solid supports are delivered to the emulsion microdropletswith the cell mixtures. Then, emulsion microdroplets are sorted intothree different incubation receptacles. The receptacles are incubatedfor 2 hr, 6 hr, or 10 hr. During sorting, the RFIDs are read by an RFIDreader, and a computer is used to record the RFIDs that are associatedwith each protocol. The method enables combinatorial screens withmultiple protocols run concurrently. Different protocols can comprisedifferent media, incubation temperatures, interacting cells, drugs,proteins, or molecules, temperatures, or incubation times.

In some embodiments of the invention, cells are used to both induceresponses in other cells and to compartmentalize polynucleic acidsunique to clones, for example, a polynucleic acid barcode or a variableimmune receptor. In some embodiments, cell responses are induced by amolecular reagent affixed to a solid support, for example, a bead or amicrofluidic chamber. In some embodiments, the molecular reagent and thesolid support act as an inducer, rather than a cell. In someembodiments, the molecular reagent is expressed by filamentous phage, orother kind of virus or virus-like particle, rather than a cell or solidsupport. In some embodiments, the particle acts as an inducer, ratherthan a cell. In certain embodiments, said molecular reagent is a proteinsuch as a cytokine, or an organic drug substance.

In some embodiments, microbial cells, such as recombinantly engineeredyeast are used as inducer cells. For example, yeast display methods canbe used for rapid and cheap expression of TCRs and antibody fragments(scFv). In some embodiments, tailed-end PCR is used to add polynucleicacid “adapters” to the heavy and light chain linkage amplicons, forhomologous recombination in vivo. The modified DNA libraries can be thenelectroporated into Saccharomyces cerevisiae cells with a linearizedvector (pYD) that contains a GAL1/10 promoter and an Aga2 cell walltether. The GAL1/10 promoter induces expression of the scFv protein inmedium that contains galactose. The Aga2 cell wall tether can be used toshuttle the scFv to the yeast cell surface and tether the scFv to theextracellular space. Transformed cells can be then expanded and inducedwith galactose. The scFv-expressing yeast library is then used as alibrary of inducer clones.

3) High-Throughput Functional Analysis

Libraries of clonal cells, prepared by any of the methods above, can becharacterized and quantified through bulk sequencing. Prior toperforming any kind of functional assays, it can be useful tocharacterize and quantify the contents of a population of clones. Forexample, methods that generate populations of clones can compriseseveral technical steps, which can yield inadequate results from time totime, and thus deep sequencing can be performed as quality control. RNAcan be isolated from a population of clonal cells, and then subjected toRT-PCR to make libraries of DNA for bulk sequencing. If the librarycomprises antibodies or TCRs, RT-PCR can be performed using a pool ofV-gene primers on the 5′ end of the transcripts, and C-gene primers onthe 3′ end of the transcripts. In addition to the transcript-specificsequences, the RT-PCR primers can have subsequences that comprisepolynucleic acid sequences that enable bulk sequencing (e.g., Illuminasequencing). These polynucleic acid sequences, termed sequencingadapters (e.g., Illumina sequencing adapters), enable hybridization ofthe library to bulk sequencing flow cells, such that bridgeamplification and sequencing by synthesis takes place. Similar methodscan be used for barcoded cDNA libraries, or any other RNAs that enabletrace back to single cell clones. Sequencing methods offered bycommercial providers such as Pacific Biosciences, Oxford Nanopore, andRoche have similar utility as methods offered by Illumina.

In bulk sequencing, read errors can be difficult to distinguish frombiological variation, which complicates identification of clones. Toreduce the frequency of base call errors, the expected error filteringmethod know in the art, e.g., methods of Edgar and Flyvbj erg(Bioinformatics 2015 Nov. 1; 31(21):3476-82), can be used. For example,the expected number of errors (E) for a read can be calculated from itsPhred scores. Reads with E>1 can be discarded, leaving reads for whichthe most probable number of base call errors is zero. When greatersensitivity to rare variants is needed, larger values of E may be used.As an additional quality filter, singleton reads (i.e., reads with asequence found only once) can be discarded, noting that sequencingerrors are unlikely to be reproduced by chance so that sequences foundtwo or more times have a high probability of being correct.

Methods described above can be used interchangeably for biologicalassays that measure activation or inactivation, and for biologicalassays that measure up-regulation or down-regulation of transcripts. Thebiological assays can be used to measure both up-regulation anddown-regulation of transcripts concurrently.

EXAMPLES Example 1 Functional Analysis of Fc Variants or Mutants

Therapeutic antibody drugs function by a variety of mechanisms. Twocommon mechanisms for therapeutic antibody drug function areAntibody-Dependent Cell-mediated Cytotoxicity (ADCC) and ComplementDependent Cytotoxicity (CDC). Both ADCC and CDC are mediated by theFragment Crystallizable (Fc) region of antibodies. In ADCC, the variabledomain of an antibody binds to an antigen exposed on the surface of acell. If enough antibody molecules bind to the antigen, NK cells bind tothe Fc domains via CD16, also known as Fc Receptor (FcR). In theclassical pathway for CDC, antibodies bind an antigen on a target cell'ssurface. Then, the C1 complex of the complement cascade binds to the Fcdomain of the antibody. Typically, at least six antibody molecules arerequired for C1 to bind. Binding of C1 to Fc then recruits remainingcomponents of the classical complement pathway, which form a membraneattack complex that works to rupture the target cell's cell membrane.The four major IgG isotypes (IgG1, IgG2, IgG3, and IgG4) differ in theircapacity for mediating ADCC and CDC. IgG3, IgG1, and IgG2 have thehighest to lowest ability to activate complement, respectively. IgG4does not activate complement. IgG1, IgG3, IgG4, and IgG2 have thehighest to lowest ability to bind FcR, respectively. Drug developerstherefore have interest in finding the optimal Fc for antibodycandidates. In certain situations, drug developers fuse high-affinityvariable domains to the optimal wild type Fc sequences. In othersituations, drug developers mutate wild type Fc sequences to generatelibraries of Fc variants, or Fc mutants. Conventionally, drug developerschoose optimal Fc variants by high-throughput screens for binders to FcRor C1, followed by functional analysis in 96-well plates. There is aneed in the field for high-throughput methods that screen directly forfunctional Fc variants, which removes the requirement for 96-well platefunctional analysis.

To screen functional Fc variants, a library of Fc mutants is generatedby methods known in the art (e.g., synthetic generation of polynucleicacids that are then assembled into protein-coding polynucleic acids,site-directed mutagenesis, or error-prone PCR). The library of Fcmutants is expressed recombinantly in Chinese hamster ovary (CHO) cells.The Fc mutants are fused to a membrane tether protein domain. In thisway, The Fc mutants are able to bind directly to FcR or C1, and inducecellular functions, while still bound to the cell membrane. Theresulting Fc mutant library comprises a population of clones, aplurality of which express a single Fc variant.

A plurality of clones from the library of variant Fc-expressing CHOcells are isolated with NK cells.between The ratio between Fc-expressingCHO cells and NK cells ranges between 1:10, and 1:20. NK-92 cells orprimary NK cells are used for the experiment. Other kinds of mammaliancell lines, for example CHO, HEK293, or Jurkat, engineered to expressCD16 receptors, are also tested, substituting NK cells.

The Fc-expressing CHO cells and NK cells are partitioned intoaqueous-in-oil droplets, and then incubated for 2, 4, 6, 12, 18, or 24hours in a 37° C. tissue culture incubator, such that functional Fcvariants expressed by the CHO clones bind to CD16 molecules of the NKcells, which activates the NK cells. These droplets are 20-200 μm indiameter. The droplets are then injected into a second microfluidic chipthat fuses the cell-containing droplets with droplets that contain lysismix and oligo-dT microbeads. The lysis mix contains a surfactant such asSDS, and poly(A) RNA transcripts bind to the oligo-dT microbeads.Overlap extension droplet PCR using primers specific to immunoglobulinand NK cell activation markers, for example, TNFa or IFNg, such that thepolynucleotides encoding the activation markers are linked throughhybridization to polynucleotides encoding Fc variants. Universal primersare also added to amplify any Fc variant in the library of engineeredCHO. The droplet overlap extension RT-PCR is performed by injectingbeads into aqueous-in-oil reactors, and incubating in a tube in aconventional thermal cycler. The plurality of polynucleic acidsgenerated by overlap extension RT-PCR are then subjected to bulksequencing to identify and quantify Fc sequences linked to NK cellactivation markers.

NK cell activation markers that can be used for these experiments areendogenous transcripts expressed by the NK cells or transcriptionalreporters engineered into NK cells. From this experiment, Fc variantsexpressed by CHO cells that induce a functional response in NK cells areidentified. Similar experiments are performed with neutrophils or othercells that phagocytose cells coated in complement, incubated with the Fcvariant library. The medium encapsulated with the cells includes C1 andother components of complement. Neutrophil activation transcripts arelinked by droplet overlap extension RT-PCR to Fc variant sequences. Theresulting library of linked polynucleic acid molecules can be thensubjected to bulk sequencing to identify and quantify Fc sequenceslinked to neutrophil activation markers.

Similar experiments are also performed with recombinant cells engineeredto express CD16 or other receptors, incubated with the Fc variantlibrary.

Variant Fc receptors that show optimal ADCC or CDC function are thenfused to an antibody variable domain with affinity toward a therapeutictarget of interest. The methods for cloning and purifying monoclonalantibodies are well known to those skilled in the art. These monoclonalantibodies are then further validated for ADCC or CDC by conventionalwell plate assays. The pharmacokinetic properties of the Fc variant areinvestigated. In many therapeutic modalities, increased antibodyhalf-life is desired and is increased by mutations in the Fc domain. TheFc-variant fused antibodies are subjected to efficacy analysis usingmouse models for cancer, efficacy analysis using opsonization studies orother types of efficacy analysis. This experiment provides highlyefficient Fc-variant fused antibodies.

Example 2 Functional Analysis of Memory B Cells

Many patients recover from severe disease for reasons currently unknownto science. For example, certain cancer patients respond better thanother patients to medical treatments. In another example, certainpatients respond better viral pathogens (e.g., Ebola, Zika, or influenzaA) than other patients. Other examples include bacterial pathogens andautoimmune disorders. In some cases, patients successfully recover fromsevere disease because they successfully mount an immune responseagainst the disease, e.g., T cell receptors or immunoglobulins that arepresent and active in good responding patients but not present in poorresponding patients might function by binding to relevant diseasetargets.

Memory B cells, or Bmems, are particularly useful for the discovery ofantibodies that helped an individual recover from serious disease. Oninitial stimulation by an antigen, naïve follicular B cellsdifferentiate into plasma cells and Bmems. Plasma cells mount theprimary humoral immune response to the antigen. Persistent Bmems ariseafter affinity maturation (mutation and selection with the antigen) ingerminal centers. A patient may have millions to billions of differentBmem clones from among which a drug developer may wish to discover anantibody that contributed to recovery from severe disease.Conventionally, screening for reactive Bmems involves incubating apopulation of Bmems with a fluorescently labeled target of interest, andthen flow sorting for binders. Methods for flow sorting are familiar tothose skilled in the art, and typically is performed using devicescommercially manufactured by suppliers such as BD, Sony, or BeckmanCoulter. However, such methods do not take Bmem cellular function intoaccount. Additionally, flow sorting is easiest with a soluble target,whereas many targets are best studied as recombinant proteins embeddedin cell membranes. Therefore, there is a need in the field forhigh-throughput cellular methods that could distinguish reactive fromnon-reactive Bmems, upon exposure to an antigen of interest.

To identify reactive Bmems, Bmems are extracted from the peripheralblood of a patient that has recovered from Ebola infection by flowcytometry or antibody-coated magnetic beads. The Bmems are thenincubated ex vivo with the antigen of interest (e.g., recombinantinducer cells that express a library of domains of the glycoprotein (GP)that comprises surface projections of the lipid envelope of the Ebolavirus). The incubation takes place inside aqueous-in-oil microdropletsor in nanoliter wells in a microfluidic device. The B cells aresubjected to emulsion overlap extension RT-PCR to generate a library ofpolynucleic acids that link heavy immunoglobulin sequences totranscripts indicative of Bmem cell activation. The activationtranscript can be endogenous transcripts of Bmem cells such as Ki-67 ortranscripts of a reporter engineered into the Bmem. From thisexperiment, antibodies expressed by Bmem cells that respond to theantigen are identified by the activation biomarkers, and that thesebiomarker transcripts are additionally hybridized to transcripts thatdiscriminate the presence of a GP domain on a cell co-encapsulated withthe target Bmem.

Antibody sequences linked to Bmem activation markers are then cloned andpurified as monoclonal antibody protein. The methods are performedeither on a single antibody sequence, or on a library of antibodysequences. If performed on a library of sequences are cloned andpurified, recombinant proteins expressed from the library are thenfurther screened for binding or function in vitro. The methods forcloning, purifying, and screening recombinant antibodies are well knownto those skilled in the art. Isolated monoclonal antibodies are thenvalidated for binding and function through conventional well-plateassays or mouse models. This experiments allow identification ofantibodies that helped an individual recover from Ebola infection.

Bmem response to antigens is also compared across many individuals, as amethod for identifying appropriate polypeptide sequences for developmentof broadly efficacious vaccines. For example, the immunogenic domains ofEbola GP are discovered, associated with good outcomes in patients whohave recovered from infection, and then those domains form the basis ofa vaccine that generates a protective antibody response and Bmempopulation for individuals who receive the vaccine but have never beenexposed to Ebola virus.

Similar methods are further used to find antigenic peptides for T cells.

Example 3 Functional Analysis for Discovery of Antibody Targets

Many patients recover from severe disease for reasons currently unknownto science. For example, certain cancer patients respond better thanother patients to medical treatments. In another example, certainpatients respond better viral pathogens (e.g., Ebola, Zika, or influenzaA) than other patients. Other examples include bacterial pathogens andautoimmune disorders. In some cases, patients successfully recover fromsevere disease because they successfully mount an immune responseagainst the disease, e.g., immunoglobulins that are present and activein good responding patients but not present in poor responding patientsmight function by binding to relevant disease targets.

However, because of the complexity of many diseases and the complexityof immune systems, it remains difficult to discover the immunoglobulinsand their respective targets. This knowledge would be extremely usefulto researchers studying the mechanism of disease, the mechanism ofdisease response, and methods for treating disease. For example, anantibody produced by a cancer patient binds to a tumor throughspecificity to a glycoprotein target expressed by the tumor and unknownto science. Binding of this antibody to the tumor then induces ADCC andCDC, which leads to complete remission of the cancer. However, it isdifficult to find the sequence of the functional antibody as well as thetarget of the functional antibody. Drug developers may use the antibodyas a drug, or develop closely related sequences once the endogenoussequence is known. Drug developers may also use the newly discoveredtarget to immunize mice or screen phage display libraries, and developnovel antibodies with affinity toward the newly discovered target.Conventionally, it is difficult and expensive to obtain the completecomplement of glycoprotein targets present in a tumor. Therefore, thefield would benefit from a high-throughput method that identifies theantibody and its target, using the glycoprotein targets expressed by thetumor and the immune repertoire sequences expressed by the patient. Themethod is not limited to cancer, and can be applied to any disease thatinvolves the immune system.

To identify an antibody and its target using the glycoprotein targetsexpressed by the tumor and the immune repertoire sequences expressed bythe patient, B cells are isolated from a cancer patient, for example,peripheral blood, bone marrow, or tumor infiltrating lymphocytes. Thecancer patient recently recovered from the cancer, is currently fightingthe cancer, or is fighting the cancer and receiving immune modulatingtherapies. Methods for separating B cells from non-B cells include flowcytometry and antibody-coated magnetic beads. B cells incubated with anantigen, pool of antigens, cells, or tissues of interest (e.g., a tumoror tumor cells) are used for the purpose of activating or expanding Bcells of interest to the study. The B cells are subjected to emulsionoverlap extension RT-PCR to generate a library of polynucleic acids withnatively linked heavy and light chain immunoglobulin pairings. Theselibraries of immunoglobulins are then used to engineer recombinantantibody-secreting cells, for example, Chinese hamster ovary cells.Methods for engineering cells are familiar to those skilled in the art,and may include electroporation of plasmids, lentiviral transduction,lipid-based transfection or transient transfection of a plasmid. PrimaryB cells are used to generate antibody-secreting hybridomas.

A library of cell clones secreting antibodies is screened against alibrary of cell clones expressing putative antibody targets. Theantibody targets are encoded by complementary DNA cloned into anexpression plasmid. The cDNAs are derived from RNA isolated from atumor, for example, a tumor that was surgically removed from the patientthat provided the sample of B cells, or from a different patient orpatients. The tumor is the same tissue of origin as the tumor from thepatient that provided the sample of B cells, or from a different tissueof origin as the tumor from the patient that provided the sample of Bcells. cDNA derived from tissues unrelated to tumors, or human donorswithout cancer is used. For some experiments, the library of putativeantibody targets generated by engineering recombinant cells withsynthetic DNA cloned into an expression plasmid is used.

A plurality of clones from the library of antibody-secreting CHO cellsare then isolated with cells that express cDNA from a matched tumor(“target clones”). A plurality of NK cells (intermediary cells) are alsoisolated with the antibody-expressing clones and the cDNA-expressingclones. A typical ratio of antibody-expressing cells to cDNA-expressingcells to NK cells is 1:1:10, or 1:1:20. NK cells comprise NK-92 cells orprimary NK cells. The cells are partitioned into aqueous-in-oildroplets, and then incubated for 2, 4, 6, 12, 18, or 24 hours in a 37°C. tissue culture incubator, such that antibodies secreted from CHOclones bind to the cDNA-expressing cells, which activates the NK cells.These droplets are 20-200 μm in diameter. The droplets are then injectedinto a second microfluidic chip that fuses the cell-containing dropletswith droplets that contain lysis mix and oligo-dT microbeads. The cellsare lysed with a surfactant such as SDS, and poly(A) RNA transcriptsbind to the oligo-dT microbeads. Overlap extension droplet PCR usingprimers specific to immunoglobulin and NK cell activation markers,(e.g., endogenous transcripts of NK cells such as TNFa or IFNg, ortranscripts of reporters engineered into NK cells), such that thepolynucleotides encoding the activation markers are linked throughhybridization to polynucleotides encoding immunoglobulin. Immunoglobulinis also linked through hybridization to specific identifying sequencesin the putative target cDNA transcript. For example, the cDNAtranscripts of the putative targets may contain synthetic polynucleicacid barcodes or unique non-synthetic sequences. Droplet overlapextension RT-PCR is performed by injecting the beads into aqueous-in-oilreactors, and incubating in a tube in a conventional thermal cycler. Theplurality of polynucleic acids generated by overlap extension RT-PCR arethen subjected to bulk sequencing to identify and quantify antibodysequences linked to NK cell activation markers, and then link theseantibody sequences to putative cDNA target transcripts. Heavy chainimmunoglobulin is linked to activations markers and light chainimmunoglobulin, to form fusion complexes of three, four, or moretranscripts such that polynucleic acid sequences sufficient to produceantibody protein are generated. Heavy chain immunoglobulin is linked toactivations markers and light chain immunoglobulin, such that only twotranscripts are linked, for example, heavy chain immunoglobulin andTNFα. From this experiment, antibodies secreted by antibody-secretingCHO cells that induce a functional response in NK cells are identified,and these antibodies are linked in parallel to putative target cDNAtranscripts. In this way, an antibody is paired with its target throughhigh-throughput functional analysis.

Similar experiments are performed with libraries of antibodies that arenot derived from human repertoires. For example, antibody sequencesrandomly or synthetically generated are used. Cells that express suchlibraries comprise recombinant Chinese hamster ovary cells engineeredwith synthetically generated antibodies. The library of antibodies isthen screened against a library of recombinant cells expressing tumorcDNAs. A single monoclonal antibody is screened against a library ofrecombinant cells expressing tumor cDNAs.

Similar experiments are performed with recombinant CD16-engineered cellsinstead of NK cells. Recombinant CD16-engineered cells also express areporter transcript, which is used as an activation biomarker.Similarly, any cell reactive to antibodies binding to a cell surface isused instead of NK cells.

Antibody sequences linked to NK cell activation markers are then clonedand purified as monoclonal antibody protein. A cDNA target linked to NKcell activation and at least one antibody sequence from an immunerepertoire is then used to discover novel antibodies against the cDNAtarget, for example, through mouse immunization, phage display, or yeastdisplay. The methods for cloning and purifying monoclonal antibodies arewell known to those skilled in the art. In parallel, the associatedtarget cDNA is cloned and used to validate the monoclonal antibody byconventional well plate assays or mouse models for cancer.

Example 4 Functional Screen of Therapeutic Antibody Candidates

Therapeutic antibody drugs function by a variety of mechanisms, butthose skilled in the art of antibody drug development would appreciatethat the ability of an antibody to bind to a given target does notnecessarily guarantee that the antibody induces the required biologicalfunction. For example, proteins expressed on the surface of immune cellsthat modulate cancer (e.g., PD-1, OX-40, or LAG3) may be immuneactivators or immune repressors. A drug developer looks for drugs thatagonize or antagonize immune activators or immune repressors. Forexample, the putative therapeutic mechanism of an anti-OX40 antibody isto act as an agonist. OX40 is expressed on the surface of T cells, andbinding of OX40L activates T cells. Activated T cells then can mount animmune response against the tumor, which improves the condition of thepatient. In certain therapeutic modalities, activating OX40 occurs bycrosslinking several molecules of OX40, which then induces a signaltransduction cascade inside of the cell. For example, TRAF2, 3, and 5,and PI3K are activated upon OX40L binding to an OX40-expressing T cell.Certain antibodies that bind to OX40 mimic the functional effect ofOX40L, however, other antibodies that bind to OX40 do not mimic thefunctional effect of OX40L. Though there are many high throughputmethods that one skilled in the art uses to identify binders to thetarget of interest (e.g., phage display, yeast display, hybridomascreening, etc.), methods for identification of antibodies that induce aspecific biological functional remain low-throughput, for example,practically limited to no more than 10-100 assays per week perlaboratory technician. Therefore, there is a need for high-throughputmethods to identify binders that induce a specific biological function.For example, high-throughput methods provided herein are used toidentify immune agonists or antagonists, or to identify activation ofsignal transduction cascades.

To identify binders that induce a specific biological function, a mouseis immunized with a target protein of interest in the field of cancerbiology. The target is a protein that is overexpressed on the surface oftumor cells (e.g., CD20, Her2, or EGFR), or a protein expressed on thesurface of immune cells that modulate cancer (e.g., PD-1, OX40, orLAG3). Typical wild type mouse strains include BL/6, SJ/L, and Balb/c.The genome of the mouse has been engineered to express fully human orchimeric antibodies, for example, the Medarex or Trianni mice. Beforesacrificing the animal, serum is removed and assessed for titer againstthe target of interest. Lymph nodes are then removed from the mouse.Spleens and bone marrow are removed from the mouse. Single cellsuspensions are then generated from the organs, and B cells areseparated from non-B cells. Methods for generating single cellsuspensions from mouse organs include enzymatic digestion and physicaldisaggregation. Methods for separating B cells from non-B cells includeflow cytometry and antibody-coated magnetic beads.

Specifically, OX40 is used as the immunogen for mouse immunization.Mouse immunization, overlap extension RT-PCR, and CHO cell engineeringare used to generate a library of CHO cells that secrete antibodycandidates against OX40. These antibodies are pre-enriched for bindersagainst OX40, for example through scFv yeast or phage display. Aplurality of clones from the library of antibody-secreting CHO cells arethen isolated with OX40 expressing cells, for example, primary T cellsor Jurkat cells engineered with OX40. The cells are partitioned intoaqueous-in-oil droplets, and then incubated for 2, 4, 6, 12, 18, or 24hours in a 37° C. tissue culture incubator. These droplets are 20-200 μmin diameter. The droplets are then injected into a second microfluidicchip that fuses the cell-containing droplets with droplets that containlysis mix and oligo-dT microbeads. Cells are lysed with a surfactantsuch as SDS, and poly(A) RNA transcripts bind to the oligo-dTmicrobeads. Overlap extension droplet PCR using primers specific toimmunoglobulin and T cell activation markers, (e.g., endogenoustranscripts of T cell such as CD69 and IFNg or transcripts of a reporterengineered into target cells), such that the polynucleotides encodingthe activation markers are linked through hybridization topolynucleotides encoding immunoglobulin. Droplet overlap extensionRT-PCR is performed by injecting the beads into aqueous-in-oil reactors,and incubating in a tube in a conventional thermal cycler. The pluralityof polynucleic acids generated by overlap extension RT-PCR are thensubjected to bulk sequencing to identify and quantify antibody sequenceslinked to T cell activation markers. Heavy chain immunoglobulin islinked to activations markers and light chain immunoglobulin, to formfusion complexes of three, four, or more transcripts such thatpolynucleic acid sequences sufficient to produce antibody protein aregenerated. Heavy chain immunoglobulin is linked to activations markersand light chain immunoglobulin, such that two transcripts are linked,for example, heavy chain immunoglobulin and CD69. The antibody sequenceis linked to the full transcriptome, and then the transcriptome isanalyzed bioinformatically to detect sequence changes indicative ofchanges in cell function. From this experiment, antibodies secreted byantibody-secreting CHO cells that induce a functional response in Tcells are identified.

Antibody sequences linked to T cell activation markers are then clonedand purified as monoclonal antibody protein. The methods for cloning andpurifying monoclonal antibodies are well known to those skilled in theart. These monoclonal antibodies are then validated for T cellactivation by conventional well plate assays or mouse models for cancer.For example, NOD SCID gamma (NSG) mice are grafted with human immunecell progenitors, which give rise to differentiated human T cells in themice. NSG mice are provided by commercial vendors such as Jackson Labs.The mice are then grafted with tumor cells, and provided with thecandidate monoclonal antibody. The response of the T cells in theseconditions is then compared to a variety of controls, for example, NSGmice with differentiated human T cells and tumor cells, but no antibody.

Example 5 Epitope Characterization Using Massively Parallel FunctionalAnalysis

Antibodies can be discovered by screening for binders against a completeprotein, or a domain of a protein that comprises at least 100 aminoacids, for example, through immunization of a mouse or panning with aphage display library. A drug developer is often interested tocharacterize the specific binding epitope of an antibody of interest.This information is useful for government regulatory filings but alsomay be useful for choosing antibodies with a desired functional profile,for example, antagonism or agonism of a protein or pathway. However,epitope characterization is conventionally a slow and expensive process.Additionally, conventional methods for epitope characterization do nottake cellular function into account, rather, the conventional methodsonly take binding affinity into account. The field would benefit from ahigh-throughput epitope screening method that is based on functionalanalysis.

For a high-throughput epitope screening, an anti-Her2 antibody isgenerated by immunizing a mouse with the soluble, complete extracellulardomain of Her2 and a library of putative Her2 epitopes is generated byengineering recombinant cells with peptides or domains from Her2,representing 10, 50, 100, 150, 200, or 250 amino acids, tethered to thecell membrane with a transmembrane domain. The library of Her2 epitopescomprises a set of overlapping peptides or domains that tile across thecomplete extracellular domain of the Her2 protein. The mRNA transcriptencoding the epitope target also comprises a nucleic acid barcodesequence flanked by universal priming sites. The universal priming sitesare used to amplify the nucleic acid barcode, which is used to identifythe specific Her2 epitope clone. A plurality of single cells from alibrary of 5, 10, 50, 100, 150, 200, or 1000 epitope-expressing clonesare partitioned into aqueous-in-oil droplets with NK cells and a CHOcell that secretes the anti-Her2 antibody of interest, and then the cellmixtures are incubated for 2, 4, 6, 12, 18, or 24 hours in a 37° C.tissue culture incubator. If the antibody binds to a given epitope, thenthe antibodies coating the epitope-expressing cell bind to CD16molecules of the NK cells, which activates the NK cells. These dropletsare 20-200 μm in diameter. The droplets are then injected into a secondmicrofluidic chip that fuses the cell-containing droplets with dropletsthat contain lysis mix and oligo-dT microbeads. Cells are lysed with asurfactant such as SDS, and poly(A) RNA transcripts bind to the oligo-dTmicrobeads. Overlap extension droplet PCR using primers specific to theepitope clone and NK cell activation markers, for example, TNFa or IFNg,such that the polynucleotides encoding the activation markers are linkedthrough hybridization to polynucleotides encoding the Her2 epitope. TheNK cells can be NK-92 cells or primary NK cells or other kinds ofmammalian cell lines, for example CHO, HEK293, or Jurkat, engineered toexpress CD16 receptors, where the artificial reporter substitutesendogenous NK activation markers. Universal primers are also used toamplify an epitope in the library of engineered epitopetarget-expressing cells. Droplet overlap extension RT-PCR is performedby injecting the beads into aqueous-in-oil reactors, and incubating in atube in a conventional thermal cycler. The plurality of polynucleicacids generated by overlap extension RT-PCR are then subjected to bulksequencing to identify and quantify Her2 epitope clone sequences linkedto NK cell activation markers. From this experiment,Her2 epitopes thatinduce a functional response in NK cells are identified. The method canbe used for any antibody that functions via ADCC.

A soluble form of the extracellular domain of OX40 is also used as animmunogen for mouse immunization. CHO cell engineering is used togenerate a CHO clone that secretes an antibody against OX40. A libraryof cell-expressed putative OX40 epitopes is generated by engineeringprimary T cells or Jurkat cells with peptides or domains from OX40,representing 10, 50, 100, 150, 200, or 250 amino acids, tethered to thecell membrane with a transmembrane domain. The library of OX40 epitopescomprises a set of overlapping peptides or domains that tile across thecomplete extracellular domain of the OX40 protein. The mRNA transcriptencoding the epitope target also comprises a nucleic acid barcodesequence flanked by universal priming sites. The universal priming sitesare used to amplify the nucleic acid barcode, which is used to identifythe OX40 epitope clone.A plurality of single cells from a library of 5,10, 50, 100, 150, 200, or 1000 epitope-expressing clones are partitionedinto aqueous-in-oil droplets with NK cells and a CHO cell that secretesthe anti-OX40 antibody of interest, and then the cell mixtures areincubated for 2, 4, 6, 12, 18, or 24 hours in a 37° C. tissue cultureincubator. These droplets are 20-200 μm in diameter. The droplets arethen injected into a second microfluidic chip that fuses thecell-containing droplets with droplets that contain lysis mix andoligo-dT microbeads. The cells are lysed with a surfactant such as SDS,and poly(A) RNA transcripts bind to the oligo-dT microbeads. Overlapextension droplet PCR using primers specific to the OX40 epitopes and Tcell activation markers, for example, CD69 and IFNg, such that thepolynucleotides encoding the activation markers are linked throughhybridization to polynucleotides encoding an OX40 epitope. When thetarget cells are engineered to comprise a reporter gene by introductionof a plasmid or genome engineering, the reporter transcripts are used asactivation markers. Droplet overlap extension RT-PCR is performed byinjecting the beads into aqueous-in-oil reactors, and incubating in atube in a conventional thermal cycler. The plurality of polynucleicacids generated by overlap extension RT-PCR are then subjected to bulksequencing to identify and quantify antibody sequences linked to T cellactivation markers. In this way, epitopes necessary and/or sufficientfor OX40 activation are discovered. The epitope sequence is linked tothe full transcriptome, and then the transcriptome is analyzedbioinformatically to detect sequence changes indicative of changes incell function. From this experiment, the OX40 epitopes that induce afunctional response in T cells, in the presence of the anti-OX40antibody of interest, are identified. The method can be used for anyantibody drug that functions via checkpoint inhibition.

Similar methods are used to characterize the functional binding epitopesof an antibody which is known to induce functional transcriptionalchanges in another type of cell. Candidate antibodies are cloned andpurified as monoclonal antibody protein. The methods for cloning andpurifying monoclonal antibodies are well known to those skilled in theart. These monoclonal antibodies are then validated for cell activationby conventional well plate assays or mouse models for cancer. Forexample, NOD SCID gamma (NSG) mice are grafted with human immune cellprogenitors, which give rise to differentiated human T cells in themice. NSG mice are provided by commercial vendors such as Jackson Labs.The mice are then grafted with tumor cells, and provided with thecandidate monoclonal antibody. The response of the T cells in theseconditions is then compared to a variety of controls, for example, NSGmice with differentiated human T cells and tumor cells, but no antibody.

Newly discovered epitopes that are necessary and sufficient to inducecell function, when paired with a given antibody, are then used todiscover new antibodies that comprise similar or better functionality.

Example 6 Discovery of Bispecific Drugs

In many therapeutic situations, it is desirable for a single molecule tobind to two different targets, thereby inducing two differenttherapeutic mechanisms independently. For example, one component of thedrug is an antibody fragment that binds one target, and anothercomponent of the drug is an antibody fragment that binds a secondtarget. There are many formats for such bispecific drugs, for example,“bis-scFv”, wherein two different scFv sequences, with two differentspecificities, are fused together with a peptide linker. For example,one scFv binds to and agonizes CD3, and the second scFv binds to EGFR,which is often over-expressed on the surface of certain tumors. Agonismof CD3 activates T cells, which then have tumor killing activity.Bispecific drugs are not limited to antibodies, for example, two TCRscan be fused to generate a bispecific TCR, an antibody can be fused to aTCR, or a recombinant ligand can be fused to an antibody fragment (e.g.,OX40L fused to anti-CD3 antibody). A fusion molecule whose individualparts generate individual activities may not necessarily generate bothactivities when the individual parts are fused. Conventionally,bispecific activities are screened at a throughput of no more than10-100 candidates per week per laboratory technician. Therefore, thereis a need in the field for high-throughput methods that screen formultiple biological functions simultaneously.

To screen multiple biological functions simultaneously, libraries ofbispecific drug candidates are subjected to the screening procedures ofthe present invention. Specifically, NK cell activation screens areperformed with two distinct antibody targets in parallel (e.g., CD3 andEpCAM). Furthermore, NK cell activation screens are performed in serieswith TCR activation screens. Various combination of combinatorialscreens is possible with the methods of the present invention.

Example 7 Functional Screen of Therapeutic T Cell Receptor Candidates

Therapeutic TCR drug discovery comprises mining of synthetic TCRrepertoires, immunization and TCR recovery from mice, or mining ofpopulations of human lymphocytes. Therapeutic T cell receptor drugsfunction by a variety of mechanisms, but the ability of TCR to bind to agiven target does not necessarily guarantee that the TCR induces therequired biological function.

However, it remains difficult to characterize the functional activity ofT cell receptors that are known to bind to targets of interest. Forexample, a TCR is discovered from a library using MHC multimers, forexample, MHC tetramers or MHC dextramers. When this TCR is expressedrecombinantly in a T cell, the desired therapeutic mechanism of actionis for the TCR-engineered T cell to bind to a peptide:MHC target on, forexample, a target cell in a disease state, for example, a cancerous cellor a cell infected with a virus. However, proper binding of a TCR to acognate peptide:MHC does not necessarily guarantee that the T cell willbe activated. Therefore, the field would benefit from a method thatscreens libraries of TCRs for functional activity in the context of atarget peptide:MHC of interest. Drug developers may use the TCR as asoluble drug or TCR-engineered T cell, or develop closely related,higher-affinity, or higher-activity, sequences once a functionalsequence is known.

To screen a library of TCRs for functional activity, T cells areisolated from a cancer patient, for example, peripheral blood, bonemarrow, or TILs. The cancer patient recently recovered from the cancer,is currently fighting the cancer, or is fighting the cancer andreceiving immune modulating therapies. T cells are separated from non-Tcells using methods known in the art such as flow cytometry andantibody-coated magnetic beads. The T cells are incubated with anantigen expressed in an APC, for the purpose of activating or expandingT cells of interest to the study. Primary T cells are subjected toemulsion overlap extension RT-PCR to generate a library of polynucleicacids with natively linked TCRαβ pairings. These libraries of TCRs arethen used to engineer recombinant TCR-expressing cells, for example,Jurkat cells. Alternatively, the TCRαβ library is generatedsynthetically using molecular biology, instead of being derived fromnatural TCRαβ sequences expressed by primary T cells. Methods forengineering of recombinant cells can include electroporation ofplasmids, lentiviral transduction, and lipid-based transfection. Cellstransiently transfected with plasmids that express TCRs, or mRNAs thatencode the TCRs of interest, primary T cells that express TCRs, orprimary T cells engineered to express recombinant TCRs are used as theTCR-expressing cells.

A plurality of clones from the library of TCR-engineered cells are thenisolated with the cells that express a cDNA, or cells from a tissue ofinterest, or cells expressing a tandem minigene (“target-expressingclones”). cDNAs are cloned into expression vectors that includepolynucleotide sequences that encode for MHC expression, for example,HLA A*02:01, HLA A*24:02, or HLA DPB*04:01. This enables peptide targetpresentation in human antigen presenting cells that do not express theMHC of interest, or non-human antigen presenting cells. The APCs arecell lines, such as HEK293 or CHO cells, or primary cells, such asdendritic cells or B cells.

A plurality of clones from the library of TCR-engineered cells are thenisolated with the target-expressing clones. The ratio of TCR-expressingcells to target-expressing cells is 1:1, 10:1, or 1:10. The cells arepartitioned into aqueous-in-oil droplets, and then incubated for 2, 4,6, 12, 18, or 24 hours in a 37° C. tissue culture incubator, such thatthe TCR-expressing clones bind to the cDNA-expressing cells, whichactivates the T cells. These droplets are 20-200 μm in diameter. Thedroplets are then injected into a second microfluidic chip that fusesthe cell-containing droplets with droplets that contain lysis mix andoligo-dT microbeads. The cells are lysed with a surfactant such as SDS,and poly(A) RNA transcripts bind to the oligo-dT microbeads. Overlapextension droplet PCR using primers specific to the target barcode ortarget sequence, and T cell activation markers, for example, CD69 orIFNg, such that the polynucleotides encoding the activation markers arelinked through hybridization to polynucleotides that identify the targetclone. TCR sequences from the T cells are also linked throughhybridization to specific identifying sequences in the target cDNAtranscript. The cDNA transcripts of the putative targets may containsynthetic polynucleic acid barcodes or unique non-synthetic sequences.Droplet overlap extension RT-PCR is performed by injecting the RNA-boundbeads into aqueous-in-oil reactors, and incubating in a tube in aconventional thermal cycler. The T cell activation markers areendogenous transcripts expressed by the T cells, or transcriptionalreporters engineered into T cells. The plurality of polynucleic acidsgenerated by overlap extension RT-PCR are then subjected to bulksequencing to identify and quantify TCR sequences linked to T cellactivation markers, and then link these TCRβ to putative cDNA targettranscripts. TCRβ is linked to T cell activations markers and TCRα, toform fusion complexes of three, four, or more transcripts such thatpolynucleic acid sequences sufficient to produce TCR protein aregenerated. TCRβ is linked to T cell activation markers and TCRα, suchthat only two transcripts are linked in a single molecule, for example,TCRβ and CD69. If the activation biomarkers are not activated, feweroverlap extension RT-PCR products will be generated, or no products willbe generated, depending on the background expression level of theactivation biomarker. From this experiment, cognate pairings between thepeptide:MHC of interest and the TCRs from the TCR library that induce afunctional response in T cells are identified. In this way, thousands,tens of thousands, hundreds of thousands, or millions of TCRs arediscovered through high-throughput functional analysis. Polynucleicacids comprising the peptide:MHC target are linked to the fulltranscriptome of the T cells, and then the transcriptome is analyzedbioinformatically to detect sequence changes indicative of changes incell function.

TCR sequences linked to T cell activation markers are then re-engineeredinto soluble format and purified as protein. The methods for cloning andpurifying monoclonal TCRs are well known to those skilled in the art. Inparallel, the associated target cDNA is cloned and used to validate theTCR by conventional well plate assays or mouse models for cancer. TheTCR is engineered into T cells and used as a therapy, for example,adoptive T cell cancer therapy. The TCR-engineered T cells are validatednon-clinically using in vitro methods, such as cell killing assays, forexample by quantifying tumor cell killing by the TCR-engineered T cellsin vitro. The TCR-engineered T cells are further validated with a mousemodel, for example, NSG mice grafted with human lymphocytes, theTCR-engineered T cells, and tumor cells, wherein tumor cell killing ismeasured in vivo.

Libraries of TCRs not derived from human repertoires or randomly orsynthetically generated can be used. When the target sequence is linkedto the full transcriptome, the transcriptome is analyzedbioinformatically to detect sequence changes indicative of changes incell function.

Example 8 Functional Analysis for Discovery of T Cell Receptor Targets

Because of the complexity of many diseases and the complexity of immunesystems, it remains difficult to discover natural T cell receptors andtheir respective targets. This knowledge would be extremely useful toresearchers studying the mechanism of disease, the mechanism of diseaseresponse, and methods for treating disease. For example, a TCR producedby a cancer patient binds to a tumor through specificity to apeptide:MHC target expressed by the tumor and unknown to science.Binding of the TCR to the tumor then induces cytotoxicity, clonepropagation, and stimulation of other immune cells, which leads tocomplete remission of the cancer. One skilled in the art can appreciatethe difficulty of finding the sequence of the functional TCRs well asthe peptide:MHC target of the functional TCR. Drug developers may usethe TCR as a soluble drug or TCR-engineered T cell, or develop closelyrelated sequences once the endogenous sequence is known. Conventionally,it is difficult and expensive to obtain the complete complement ofpeptide:MHC targets present in a tumor. Therefore, the field wouldbenefit from a high-throughput method that identifies the TCR and itspeptide:MHC target, using the glycoprotein targets expressed by thetumor and the immune repertoire sequences expressed by the patient. Themethod is not limited to cancer, and can be applied to any disease thatinvolves the immune system.

To identify TCR and its peptide:MHC target, T cells are isolated from acancer patient, for example, peripheral blood, bone marrow, or TILs. Insome embodiments of the invention, the cancer patient recently recoveredfrom the cancer, is currently fighting the cancer, or is fighting thecancer and receiving immune modulating therapies. T cells are separatedfrom non-T cells by methods such as flow cytometry and antibody-coatedmagnetic beads. The T cells are incubated with an antigen expressed inan APC, a pool of antigens expressed as a library of APC clones, celllines, or primary tissues of interest (e.g., a tumor or tumor cells),for the purpose of activating or expanding T cells of interest to thestudy. The T cells are subjected to emulsion overlap extension RT-PCR togenerate a library of polynucleic acids with natively linked TCRabpairings. These libraries of TCRs are then used to engineer recombinantTCR-expressing cells, for example, Jurkat cells. Cells are engineeredusing methods known in the art, such as electroporation of plasmids,lentiviral transduction, and lipid-based transfection. Recombinant cellstransiently transfected with plasmids that express TCRs, or mRNAs thatencode the TCRs of interest, The TCR-expressing cells are primary Tcells that express TCRs, or primary T cells engineered to expressrecombinant TCRs.

A library of cell clones engineered to express surface TCRs is screenedagainst a library of cell clones expressing putative TCR targets.Targets are encoded by complementary DNA cloned into an expressionplasmid or a lentivirus. The cDNAs are derived from RNA isolated from atumor, for example, a tumor that was surgically removed from the patientthat provided the sample of T cells, or from a different patient orpatients. The cDNAs are cloned into expression vectors that includepolynucleotide sequences that encode for MHC expression, for example,HLA A*02:01, HLA A*24:02, or HLA DPB*04:01. This enables peptide targetpresentation in human antigen presenting cells that do not express theMHC of interest, or non-human antigen presenting cells. The APCs arecell lines, such as HEK293 or CHO cells or primary cells, such asdendritic cells or B cells. MHC and the target cDNA are encoded on asingle mRNA molecule, which also comprises a nucleic acid barcodesequence flanked by universal priming sites. The universal priming sitesare used to amplify the nucleic acid barcode, which is used to identifythe cDNA clone. The tumor is the same tissue of origin as the tumor fromthe patient that provided the sample of T cells, or from a differenttissue of origin as the tumor from the patient that provided the sampleof T cells. The cDNA is derived from tissues unrelated to tumors, orhuman donors without cancer. The library of putative TCR targets isgenerated by engineering recombinant cells with synthetic DNA clonedinto an expression plasmid.

A plurality of clones from the library of TCR-engineered cells are thenisolated with the cells that express a library of cDNAs(“target-expressing clones”). A typical ratio of TCR-expressing cells totarget-expressing cells 1:1, 10:1, or 1:10. The cells are partitionedinto aqueous-in-oil droplets, and then incubated for 2, 4, 6, 12, 18, or24 hours in a 37° C. tissue culture incubator, such that theTCR-expressing clones bind to the cDNA-expressing cells, which activatesthe T cells. These droplets are 20-200 μm in diameter. The droplets arethen injected into a second microfluidic chip that fuses thecell-containing droplets with droplets that contain lysis mix andoligo-dT microbeads. The cells are lysed with a surfactant such as SDS,and poly(A) RNA transcripts bind to the oligo-dT microbeads. Overlapextension droplet PCR using primers specific to the target barcode ortarget sequence, and T cell activation markers, for example, CD69 orIFNg, such that the polynucleotides encoding the activation markers arelinked through hybridization to polynucleotides that identify the targetclone. TCR sequences from the T cells are also linked throughhybridization to specific identifying sequences in the putative targetcDNA transcript. The cDNA transcripts of the putative targets containsynthetic polynucleic acid barcodes or unique non-synthetic sequences.Droplet overlap extension RT-PCR is performed by injecting the RNA-boundbeads into aqueous-in-oil reactors, and incubating in a tube in aconventional thermal cycler. The T cell activation markers used in theseexperiments are endogenous transcripts expressed by the T cells ortranscriptional reporters engineered into T cells. The plurality ofpolynucleic acids generated by overlap extension RT-PCR are thensubjected to bulk sequencing to identify and quantify TCR sequenceslinked to T cell activation markers, and then link these TCRβ toputative cDNA target transcripts. TCRβ is linked to T cell activationsmarkers and TCRα, to form fusion complexes of three, four, or moretranscripts such that polynucleic acid sequences sufficient to produceTCR protein are generated. TCRβ is linked to T cell activations markersand TCRα, such that only two transcripts are linked in a singlemolecule, for example, TCRβ and CD69. From this experiment, cognatepairings between peptide:MHC and TCRs that induce a functional responsein T cells are identified, and these TCRs are linked in parallel toputative target cDNA transcripts. In this way, thousands, tens ofthousands, hundreds of thousands, or millions of TCRs are paired withtheir target through high-throughput functional analysis. Whenpolynucleic acids comprising the peptide:WIC target are linked to thefull transcriptome of the T cells, the transcriptome is analyzedbioinformatically to detect sequence changes indicative of changes incell function.

Libraries of TCRs which are not derived from human repertoires or TCRsequences which are randomly or synthetically generated can be used. Thelibrary of TCRs is screened against a library of recombinant cellsexpressing tumor cDNAs. A single monoclonal T cell population is alsoscreened against a library of recombinant cells expressing tumor cDNAs.

TCR sequences linked to T cell activation markers are then re-engineeredinto soluble format and purified as protein. A cDNA target linked to Tcell activation and at least one TCR sequence from an immune repertoireis then used to discover novel TCRs against the cDNA target, forexample, through mouse immunization, phage display, or yeast display.The methods for cloning and purifying monoclonal TCRs are well known tothose skilled in the art. In parallel, the associated target cDNA iscloned and used to validate the TCR by conventional well plate assays ormouse models for cancer. The TCR is engineered into autologous T cellsand used as a therapy, for example, adoptive T cell cancer therapy. TheTCR-engineered T cells are validated non-clinically using in vitromethods, such as cell killing assays, for example by quantifying tumorcell killing by the TCR-engineered T cells in vitro. The TCR-engineeredT cells are further validated with a mouse model, for example, NSG micegrafted with human lymphocytes, the TCR-engineered T cells, and tumorcells, wherein tumor cell killing is measured in vivo.

Example 9 Functional Analysis of Tumor Infiltrating Lymphocytes

Tumor infiltrating lymphocytes (TILs) are T cells that have infiltrateda tumor in situ, and therefore are considered a rich source oftumor-antigen reactive T cells. TILs are expanded from tumor samples exvivo, to produce billions of TILs in culture. The TILs are then infusedback into the patient as a cellular therapy for combating cancer.Expansion protocols involve culture for several months with growthfactors and cytokines, which sometimes leads to efficacious cells but atother times leads to cells without efficacy. Thus, it would be useful totest the efficacy of TILs prior to infusion into the patient.

To test the efficacy of TILs, TILs are co-cultured, as the target cells,with cells that express peptide:MHC of clinical relevance, as theinducer cells. TILs are screened against a library of cell clonesexpressing tumor antigens of interest for quality control. The targetcells include peptide:MHC sequence similarity with the therapeuticallyrelevant peptide:MHC target or complementary DNA cloned into anexpression plasmid or a lentivirus. The cDNAs are derived from RNAisolated from a tumor, for example, a tumor that was surgically removedfrom the patient that provided the sample of T cells, or from adifferent patient or patients. The cDNAs are cloned into expressionvectors that include polynucleotide sequences that encode for MHCexpression, for example, HLA A*02:01, HLA A*24:02, or HLA DPB*04:01.This enables peptide target presentation in human APCs that do notexpress the MHC of interest, or non-human APCs. Cell lines, such asHEK293 or CHO cells or primary cells, such as dendritic cells or B cellsare used as the APCs. An MHC and a target cDNA are encoded on a singlemRNA molecule, which also comprises a nucleic acid barcode sequenceflanked by universal priming sites. The universal priming sites are usedto amplify the nucleic acid barcode, which is used to identify the cDNAclone. The barcode amplicons are then linked through OE-RT-PCR toinduced transcripts or TCRs.

TIL cultures that fail to demonstrate efficacy are not infused back intothe patient. Where possible, the TIL cultures may be further culturedunder different conditions, for example, in the presence of astimulatory antigen of clinical relevance to the patient.

Example 10 Functional Analysis of T Cells in Response to Drugs

Dysregulation of T cell immunity is a hallmark of many kinds of humandisease, including cancer and autoimmunity. Stimulation and suppressionof T cell immunity involves a complex interplay among a variety ofproteins, for example, LAG-3, OX40, OX40L, PD1, PDL1, TIM3, CTLA4, CD47,4-1BB, GITR, ICOS, and many others. One skilled in the art canappreciate that the field of immunology may not yet fully understand thecomplex interplay that results in stimulation and suppression of T cellimmunity. It is likely that there are many components of this complexinterplay that are unknown to science. Therefore, there remains a needfor high-throughput single cell methods for further characterization ofthe molecular mechanisms of stimulation and suppression of T cellimmunity.

To characterize the molecular mechanisms of stimulation and suppressionof T cell immunity, recombinant DNA technology is used to engineer alibrary of cells that express molecules that are known to modulateimmune regulatory pathways, such as antibodies that act as checkpointinhibitors by antagonizing molecules such as PD-1, or endogenous ligandsin immune regulatory pathways, for example, PD-L1, or secreted ormembrane-boundimmune regulatory molecules. The library of immunemodulatory cells comprises CHO, HEK293, or primary cells. Methods forengineering cells to express recombinant proteins are well known tothose skilled in the art, for example, directed genome integration,transient expression via a plasmid, or lentivirus. The library of immunemodulatory cells can comprise microbes, for example, engineeredbacteria, yeast, or filamentous phage, instead of mammalian cells. ThemRNA transcript encoding the immune modulator also comprises a nucleicacid barcode sequence flanked by universal priming sites. The universalpriming sites are used to amplify the nucleic acid barcode, which isused to identify the immune modulator clone.

The library of cells expressing recombinant immune modulators ispartitioned into aqueous-in-oil droplets with T cells, cells thatexpress checkpoint molecules, or T cells engineered to expresscheckpoint molecules, and then the cell mixture emulsions are incubatedfor 2, 4, 6, 12, 18, or 24 hours in a 37° C. tissue culture incubator.These droplets are 20-200 μm in diameter. The droplets are then injectedinto a second microfluidic chip that fuses the cell-containing dropletswith droplets that contain lysis mix and oligo-dT microbeads. The cellsare lysed with a surfactant such as SDS, and poly(A) RNA transcriptsbind to the oligo-dT microbeads. Overlap extension droplet PCR usingprimers specific to the immune modulator clone and T cell activationmarkers, for example, TNFa or IFNg, using methods described above. The Tcell activation markers comprise co-stimulatory or co-inhibitorycheckpoint molecules, such as LAG-3, OX40, OX40L, PD1, PDL1, TIM3,CTLA4, CD47, 4-1BB, GITR, or ICOS. Primers specific to the immunemodulator clone are linked to primers that amplify the full target celltranscriptome as cDNA. Bioinformatics is then used to discover genesthat were not previously implicated in immune co-stimulatory orco-inhibitory pathways, or, to further clarify the function ofpreviously characterized immune co-stimulatory or co-inhibitorypathways. Bioinformatics can be used to process the full-transcriptomedata to generate transcript expression panels of 10, 100, or 1,000 genesthat are upregulated or downregulated as part of co-stimulatory orco-inhibitory pathways. These transcript expression panels are used totest whether non-clinical candidate checkpoint inhibitor drugs have thedesired effect on T cells or other target cells. The transcriptexpression panels are also used to test whether a given cancer patientresponds to clinical-stage checkpoint molecules.

The emulsion droplet screen is further combined with FACS. For example,T cells are engineered to express a fluorescent reporter molecule thatis induced upon incubation with a co-stimulatory or co-inhibitory drug.Droplets that contain activated reporters and are therefore fluorescentare sorted using FACS. The sorted emulsion droplets that containreporter-positive cell mixtures are then processed using the methodsdescribed above. In some experiments, T cells are engineered to secretemolecules, which bind to target proteins linked to solid surfaces. Saidbinding is then detected by a method such as fluorescence resonanceenergy transfer (FRET). Droplets that bind to the target protein aretherefore fluorescent and are sorted using FACS. The sorted emulsiondroplets that contain FRET-positive cell mixtures are then processedusing the methods described above. For the experiment, a FACSmachineincorporated into microfluidic chips, or a conventional FACSmachine provided by commercial vendors such as BD or Beckman Coulter isused. Similar methods are used for identifying droplets that containantibody-secreting cells that bind to target proteins, or any other kindof cell that secretes a protein that binds a target protein. Thisprovides a population of droplets that secrete proteins that bind atarget protein. This method increases the specificity of the assay andenables to perform large combinatorial screens.

The screen benefits from performing a variety of incubation protocols inparallel. For example, mixtures of cells are incubated for 2, 4, 6, 12,18, or 24 hours in a 37° C. tissue culture incubator, followed byincubation for 2 hours at 20° C., 25° C., 30° C., 35° C., or 40° C., allin a single experiment. Mixtures of cells expressing recombinant immunemodulators mixed with T cells are partitioned, using the methodsdescribed above, into emulsion microdroplets. Light-triggeredmicrotransponders, known in the art (e.g., Mandecki US 20160175801), aredelivered to the microdroplets with the cell mixtures. Similar methodsare employed using “barcodes” encoded by RFID, quantum dots,colorimetric, or other physical means. The light-triggeredmicrotransponders are then used to track delivery of cell mixtures intosix chambers, which are then incubated for 2, 4, 6, 12, 18, or 24 hoursin a 37° C. incubator. After incubation, each emulsion is then fed backinto a microtransponder reader, which tracks delivery of cell mixturesto five chambers, at 20° C., 25° C., 30° C., 35° C., or 40° C. Amicrocomputer is used to generate a database of microtransponderbarcodes and their associated protocols. In this way, six differentfirst incubation protocols are tested combinatorially with fivedifferent second incubation protocols, for a total of 30 differentcombinations. This approach can be used for any kind of combinatorialscreen.

Example 11 Functional Validation of Engineered Adoptive Cell Therapies

TCR-engineered T cells and CAR-T cells are a newer class of therapiesthat are primarily being used for cancer and infectious disease. Theengineered cells are either autologous (i.e., derived from the patient)or allogeneic (i.e., derived from an individual other than the patient).All adoptive cell therapies must be characterized functionally prior toinfusion into patients. Typically, such assays are limited to in vitrotumor cell killing assays. However, conventional assays fail to clearlyidentify specific killing of cells expressing therapeutic targets, andany off-target effects, i.e., killing of cells that should not bekilled. Methods for functional quality control of adoptive cell therapycould make such therapies safer and more efficacious, for example, bydemonstrating superiority of particular T cell transduction methods, orshowing the specificity of a TCR or CAR-T in the context of differenttypes of cells being used for engraftment, or different cell donors.

The method of present invention is used to screen cells engineered toexpress a therapeutic TCR against a library of cell clones expressingTCR targets of interest for quality control. Such targets include, forexample, targets that are known to have peptide:MHC sequence similaritywith the therapeutically relevant peptide:MHC target. Targets areencoded by complementary DNA cloned into an expression plasmid or alentivirus. The cDNAs are derived from RNA isolated from a tumor, forexample, a tumor that was surgically removed from the patient thatprovided the sample of T cells, or from a different patient or patients.The cDNAs are cloned into expression vectors that include polynucleotidesequences that encode for MHC expression, for example, HLA A*02:01, HLAA*24:02, or HLA DPB*04:01. This enables peptide target presentation inhuman antigen presenting cells that do not express the MHC of interest,or non-human antigen presenting cells. Cell lines, such as HEK293 or CHOcells or primary cells, such as dendritic cells or B cells are used asAPCs. An MHC and a target cDNA are encoded on a single mRNA molecule,which also comprises a nucleic acid barcode sequence flanked byuniversal priming sites. The universal priming sites are used to amplifythe nucleic acid barcode, which is used to identify the cDNA clone.

Cells engineered to express a therapeutic CAR-T are screened against alibrary of cell clone expressing antibody targets of interest forquality control. Such targets include, for example, surface proteintargets that are known to have sequence similarity with thetherapeutically relevant surface protein target. Targets are encoded bycomplementary DNA cloned into an expression plasmid or a lentivirus. ThecDNAs are derived from RNA isolated from a tumor, for example, anautologous tumor that was surgically removed from the patient thatprovided the sample of T cells, or from a different patient or patients.Cell lines, such as HEK293 or CHO cells or primary cells, such asdendritic cells or B cells are used as the APCs. MHC and the target cDNAare encoded on a single mRNA molecule, which also comprises a nucleicacid barcode sequence flanked by universal priming sites. The universalpriming sites are used to amplify the nucleic acid barcode, which isused to identify the cDNA clone.

The ratio between TCR-expressing cells and target-expressing cells is1:1, 10:1, or 1:10. The cell mixtures are partitioned intoaqueous-in-oil droplets, and then incubated for 2, 4, 6, 12, 18, or 24hours in a 37° C. tissue culture incubator, such that the TCR-expressingor CAR-T cells bind to the cDNA-expressing cells, which activates the Tcells. These droplets are 20-200 μm in diameter. The droplets are theninjected into a second microfluidic chip that fuses the cell-containingdroplets with droplets that contain lysis mix and oligo-dT microbeads.The cells are lysed with a surfactant such as SDS, and poly(A) RNAtranscripts bind to the oligo-dT microbeads. Overlap extension dropletPCR using primers specific to the target barcode or target sequence, andT cell activation markers, for example, CD69 or IFNg, such that thepolynucleotides encoding the activation markers are linked throughhybridization to polynucleotides that identify the target clone. TCR orCAR-T sequences from the T cells are also linked through hybridizationto specific identifying sequences in the putative target cDNAtranscript. The cDNA transcripts of the putative targets may containsynthetic polynucleic acid barcodes or unique non-synthetic sequences.Droplet overlap extension RT-PCR is performed by injecting the RNA-boundbeads into aqueous-in-oil reactors, and incubating in a tube in aconventional thermal cycler. The T cell activation markers areendogenous transcripts expressed by the T cells. The plurality ofpolynucleic acids generated by overlap extension RT-PCR are thensubjected to bulk sequencing to identify and quantify TCR or CAR-Tsequences linked to T cell activation markers, and then link these TCRβto putative cDNA target transcripts. TCRβ is linked to T cellactivations markers and TCRα, to form fusion complexes of three, four,or more transcripts such that polynucleic acid sequences sufficient toproduce antibody protein are generated. TCRβ is linked to T cellactivations markers and TCRα, such that only two transcripts are linkedin a single molecule, for example, TCRβ and CD69. From this experiment,cognate pairings between peptide:MHC and TCRs, or CAR-T and surfacetargets, that induce a functional response in T cells are identified,and these TCRs or CAR-T are linked in parallel to putative target cDNAtranscripts. The target sequence is linked to the full transcriptome,and then the transcriptome is analyzed bioinformatically to detectsequence changes indicative of changes in cell function.

The efficacy and specificity of the adoptive TCR-engineered or CAR-Tcell therapy are estimated by benchmarking the sequence counts ofon-target and off-target activation markers, respectively. Theengineered T cell activation assay is used to generate control rangesfor manufacturing a clinical therapeutic. The assay is used duringnon-clinical development of the CAR-T or TCR-engineered adoptive T celltherapy. A transcriptome-wide activation assay can be used to discovertranscripts that comprise novel biomarkers for engineered T cell safetyor efficacy.

1. A method for functional analysis of biological cells, comprising:isolating into a monodisperse emulsion microdroplet a single target cellfrom a plurality of target cell clones of a first cell type and one ormore inducer cells from a plurality of inducer cell clones of a secondcell type; incubating isolated cells in the monodisperse emulsionmicrodroplet, wherein the isolated cells comprise the single target celland the one or more inducer cells; introducing an aqueous solutioncontaining a lysis reagent into said monodisperse emulsion microdroplet,thereby inducing lysis of the isolated cells; capturing RNA releasedfrom the isolated cells on a solid surface; and generating a library ofhybridized polynucleic acids that comprise a transcript from theisolated cells, wherein the hybridized polynucleic acids are indicativeof transcriptional change in the single target cell after the step ofincubating the isolated cells.
 2. The method of claim 1, wherein saidhybridized polynucleic acids are further indicative of transcriptionalchange in the one or more inducer cells after the step of incubating theisolated cells.
 3. The method of claim 2, wherein said transcriptionalchange in the one or more inducer cells comprises increase oftranscripts of a gene by less than tenfold.
 4. The method of claim 1,wherein the plurality of target cell clones comprise more than 10,000unique cell clones, wherein each target cell clone of the plurality oftarget cell clones is genetically distinct from each other.
 5. Themethod of claim 1, wherein the plurality of inducer cell clonescomprises more than 10,000 unique cell clones, wherein each inducer cellclone of the plurality of induce cell clones is genetically distinctfrom each other.
 6. The method of claim 4, wherein genetic diversity ofthe target cell clones is created by introducing a library of nucleicacid sequences into a population of at least 100,000 cells.
 7. Themethod of claim 5, wherein genetic diversity of the inducer cell clonesis created by introducing a library of nucleic acid sequences into apopulation of at least 100,000 cells.
 8. The method of claim 1, whereinRNA capturing is performed using oligonucleotides affixed to beads,wherein each bead has a diameter less than 10 μm.
 9. The method of claim1, wherein the hybridized polynucleic acids are generated by overlapextension polymerase chain reaction.
 10. The method of claim 1, whereinthe hybridized polynucleic acids are generated by first strandsynthesis.
 11. The method of claim 1, wherein the first cell type is alibrary of cells that express T cell receptors.
 12. The method of claim1, wherein the first cell type is a library of cells that expressantibodies.
 13. The method of claim 1, wherein the first cell type is alibrary of cells that express peptide:MHC.
 14. The method of claim 1,wherein the first cell type is a library of cells that expresspolynucleic acid barcodes.
 15. The method of claim 1, wherein cells areisolated into emulsions using microfluidics.
 16. A compositioncomprising the library of hybridized polynucleic acids of claim
 1. 17.The composition of claim 16, comprising hybridized polynucleic acids ofat least 10,000 unique sequences.
 18. The composition of claim 17,comprising hybridized polynucleic acids of at least 1,000,000 uniquesequences.
 19. A method for functional analysis of a population ofcells, comprising deep sequencing of the library of hybridizedpolynucleic acids of claim
 1. 20. A composition comprising a library ofrecombinant proteins, generated from the composition of claim
 16. 21.The composition of claim 20, wherein the library of recombinant proteinscomprises T cell receptors.
 22. The composition of claim 20, wherein thelibrary of recombinant proteins comprises peptide:MHC.
 23. Thecomposition of claim 20, wherein the library of recombinant proteinscomprises antibodies.
 24. A composition comprising a first probe and asecond probe, wherein: the first probe comprises a first subsequencethat is complementary to a transcript of an inducer cell of a first celltype and a second subsequence that is complementary to at least a partof the second probe, wherein the transcript is unique to the first celltype; and the second probe comprises a third subsequence that iscomplementary to a different transcript of a target cell of a secondcell type and a fourth subsequence that is complementary to at least apart of the first probe, wherein the amount of the different transcriptchanges when the target cell is incubated with the inducer cell.
 25. Thecomposition of claim 24, wherein the transcript unique to said firstcell type encodes a T cell receptor.
 26. The composition of claim 24,wherein the transcript unique to said first cell type encodes anantibody.
 27. The composition of claim 24, wherein the transcript uniqueto said first cell type encodes a peptide:MHC.
 28. The composition ofclaim 24, wherein the transcript unique to said first cell type encodesa polynucleic acid barcode.
 29. The composition of claim 24, wherein thetranscript unique to said first cell type encodes a recombinant protein.30. A method for functional analysis of biological cells, comprising:isolating into a monodisperse emulsion microdroplet a target cell from aplurality of target cell clones of a first cell type and one or moreinducer cells from a plurality of inducer cell clones of a second celltype; incubating isolated cells in the monodisperse emulsionmicrodroplet, wherein the isolated cells comprise the single target celland the one or more inducer cells; isolating RNA from the isolatedcells; generating a library of hybridized polynucleic acids using thecomposition of claim 24; and deep sequencing the library of hybridizedpolynucleic acids.